Affinity medicant conjugate

ABSTRACT

In an embodiment of the invention, a composition for treating a cell population comprises an Affinity Medicant Conjugate (AMC). The medicant moiety can be a toxin including an acylfulvene or a drug moiety. The affinity moiety can be an antibody, a binding protein, a steroid, a lipid, a growth factor, a protein, a peptide or non peptidic. The affinity moiety can be covalently bound to the medicant via a linker. Novel linkers that can be directed to cysteine, arginine or lysine residues based on solution pH allow greater flexibility in preserving and/or generating specific epitopes in the AMC.

PRIORITY CLAIM

This application claims priority to the U.S. Provisional Application No.61/978,195 entitled “AFFINITY MEDICANT CONJUGATES”, inventors: MichaelJ. Kelner et al. filed Apr. 10, 2014, which application is hereinexpressly incorporated by reference in its entirety. This applicationherein expressly incorporates by reference the sequence listing‘MKEL-01047US7_ST25.txt’ file, with a size of 854425 bytes, created onJun. 24, 2015.

TECHNICAL FIELD

The present invention relates to compositions and methods for treatingtarget molecules including cell populations with an affinity medicantconjugate such as an antibody drug conjugate.

BACKGROUND ART

The present invention is directed to Affinity Medicant Conjugates (AMC)including acylfulvene, Illudin and Syn-Illudin based conjugates,Affinity Medicant Linker Conjugates (AMLC), antibody-drug conjugates(ADC) and medicant-linker (ML) compounds, as well as to compositions ofthe same, and to methods for their use in treating cancer, an autoimmuneto methods of using Ligand Linker Medicant (LLM) conjugates and MLcompounds in vitro, in situ, and in vivo for the detection, diagnosis ortreatment of cells and associated pathological conditions.

SUMMARY OF INVENTION

There exists a continuing need for delivery of chemotherapeutic agentsfor which tumors do not have a medicant resistant phenotype and whichinhibit tumor growth, especially solid tumor growth, and which have anadequate therapeutic index to be effective for in vivo treatment. Theantibody medicant conjugates of the present invention can have utilityin a wide range of therapeutic applications in humans as well as inanimals in general. For example, such therapeutic applications caninclude: cancer, adenocarcinoma, carcinoma, breast cancer, prostatecancer, ovarian cancer, endometrial cancer, neuroendocrine tumors,infertility, polycystic ovary syndrome, endometriosis, and precociouspuberty. For example, veterinary and agricultural applications caninclude treatment of cancer, adenocarcinoma, carcinoma, ovarian cancer,endometrial cancer, neuroendocrine tumors, and endometriosis in farmyardand/or companion animals.

The methods of this invention include administration of an effectiveamount of an antibody medicant conjugate, preferably in the form of apharmaceutical composition, to an animal in need thereof. In a furtherembodiment, pharmaceutical compositions are disclosed containing anantibody medicant conjugate of the present invention in combination witha pharmaceutically acceptable carrier.

In various embodiments of the present invention, an affinity medicantconjugate is made up of an antibody 1110 linked to an illudin1 moiety1301. Various embodiments of the invention, are directed to the methodsfor the preparation, use, and to pharmaceutical compositions containingan illudin1 moiety 1301 linked to an antibody 1110 to form an antibodymedicant conjugate (AMC). In various embodiments the compounds of thepresent invention, the AMC can have the general formula shown in FIG.3A, where the antibody 1110 is bound to a linker 1200 which is bound toan illudin1 moiety 1301. In other various embodiments of the presentinvention, the compounds of the present AMC invention can have thegeneral formula shown in FIG. 3B, where a growth factor 1120 is bound toa linker 1200 which is bound to an illudin1 moiety 1301. In variousembodiments the compounds of the present invention includestereoisomers, solvates, and pharmaceutically acceptable salts thereof,where the linker 1200 is as defined in Table X, and the illudin1 1301 isas defined below in Table XI.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, a lipid 1180, a peptide with biological activity1190, a cytokine 1195 with a traditional linker 1240 to an illudin1moiety 1301 binds to a molecule to which the antibody 1110, the growthfactor 1120, the steroid 1140, the anti-angiogenic peptide 1130, theintegrin binding peptide 1150, the glycopeptide 1170, the lipid 1180 thepeptide with biological activity 1190, or the cytokine 1195 binds anddirects the illudin1 moiety 1301 to cell populations expressing themolecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via a traditional linker 1240 to a pro-peptide 1160 is cleaved byan enzyme and thereafter binds to receptors for the peptide and directsthe illudin1 moiety 1301 to cell populations expressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a traditional linker 1240 to anilludin2 moiety 1302 binds to a molecule to which the antibody 1110, thegrowth factor 1120, the steroid 1140, the anti-angiogenic peptide 1130,the integrin binding peptide 1150, the glycopeptide 1170, the lipid1180, the peptide with biological activity 1190, or the cytokine 1195binds and directs the illudin2 moiety 1302 to cell populationsexpressing the molecule.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a traditional linker 1240 to a pro-peptide 1160 is cleaved byan enzyme 1165 and thereafter binds to receptors for the peptide anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a FSB linker (see FIG. 15) 1220 toan illudin1 moiety 1301 binds to a molecule to which the antibody 1110,the growth factor 1120, the steroid 1140, the anti-angiogenic peptide1130, the integrin binding peptide 1150, the glycopeptide 1170, thelipid 1180, the peptide with biological activity 1190, or the cytokine1195 binds and directs the illudin1 moiety 1301 to cell populationsexpressing the molecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via a FSB Linker (see FIG. 15) 1220 to a pro-peptide 1160 iscleaved by an enzyme 1165 and thereafter binds to receptors for thepeptide and directs the illudin1 moiety 1301 to cell populationsexpressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a FSB linker (see FIG. 15) 1220 toan illudin2 moiety 1302 binds to a molecule to which the antibody 1110,the growth factor 1120, the steroid 1140, the anti-angiogenic peptide1130, the integrin binding peptide 1150, the glycopeptide 1170, thelipid 1180, the peptide with biological activity 1190, or the cytokine1195 binds and directs the illudin2 moiety 1302 to cell populationsexpressing the molecule.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a FSB Linker (see FIG. 15) 1220 to a pro-peptide 1160 iscleaved by an enzyme 1165 and thereafter binds to receptors for thepeptide and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a FSB linker (see FIG. 15) 1220 to aprotein toxin 1330 binds to a molecule to which the antibody 1110, thegrowth factor 1120, the steroid 1140, the anti-angiogenic peptide 1130,the integrin binding peptide 1150, the glycopeptide 1170, the lipid1180, the peptide with biological activity 1190, or the cytokine 1195binds and directs the protein toxin 1330 to cell populations expressingthe molecule.

In an embodiment of the present invention, a protein toxin 1330 linkedvia a FSB Linker (see FIG. 15) 1220 to a pro-peptide 1160 is cleaved byan enzyme 1165 and thereafter binds to receptors for the peptide anddirects the protein toxin 1330 to cell populations expressing thereceptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a FSB linker (see FIG. 15) 1220 to amedicant 1350 binds to a molecule to which the antibody 1110, the growthfactor 1120, the steroid 1140, the anti-angiogenic peptide 1130, theintegrin binding peptide 1150, the glycopeptide 1170, the lipid 1180,the peptide with biological activity 1190, or the cytokine 1195 bindsand directs the medicant 1350 to cell populations expressing themolecule.

In an embodiment of the present invention, a medicant 1350 linked via aFSB Linker (see FIG. 15) 1220 to a pro-peptide 1160 is cleaved by anenzyme 1165 and thereafter binds to receptors for the peptide anddirects the medicant 1350 to cell populations expressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a Mal1 linker 1210 to an illudin1moiety 1301 binds to a molecule to which the antibody 1110, the growthfactor 1120, the steroid 1140, the anti-angiogenic peptide 1130, theintegrin binding peptide 1150, the glycopeptide 1170, the lipid 1180,the peptide with biological activity 1190, or the cytokine 1195 bindsand directs the illudin1 moiety 1301 to cell populations expressing themolecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via a Mal1 linker 1210 to a pro-peptide 1160 is cleaved by anenzyme 1165 and thereafter binds to receptors for the peptide anddirects the illudin1 moiety 1301 to cell populations expressing thereceptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a Mal1 linker 1210 to an illudin2moiety 1302 binds to a molecule to which the antibody 1110, the growthfactor 1120, the steroid 1140, the anti-angiogenic peptide 1130, theintegrin binding peptide 1150, the glycopeptide 1170, the lipid 1180,the peptide with biological activity 1190, or the cytokine 1195 bindsand directs the illudin2 moiety 1302 to cell populations expressing themolecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via a Mal1 linker 1210 to a pro-peptide 1160 is cleaved by anenzyme 1165 and thereafter binds to receptors for the peptide anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a Mal1 linker 1210 to a proteintoxin 1330 binds to a molecule to which the antibody 1110, the growthfactor 1120, the steroid 1140, the anti-angiogenic peptide 1130, theintegrin binding peptide 1150, the glycopeptide 1170, the lipid 1180,the peptide with biological activity 1190, or the cytokine 1195 bindsand directs the protein toxin 1330 to cell populations expressing themolecule.

In an embodiment of the present invention, a protein toxin 1330 linkedvia a Mal1 linker 1210 to a pro-peptide 1160 is cleaved by an enzyme1165 and thereafter binds to receptors for the peptide and directs theprotein toxin 1330 to cell populations expressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with a Mal1 linker 1210 to a medicant1350 binds to a molecule to which the antibody 1110, the growth factor1120, the steroid 1140, the anti-angiogenic peptide 1130, the integrinbinding peptide 1150, the glycopeptide 1170, the lipid 1180, the peptidewith biological activity 1190, or the cytokine 1195 binds and directsthe medicant 1350 to cell populations expressing the molecule.

In an embodiment of the present invention, a medicant 1350 linked via aMal1 linker 1210 to a pro-peptide 1160 is cleaved by an enzyme 1165 andthereafter binds to receptors for the peptide and directs the medicant1350 to cell populations expressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with an azlactone linker 1230 to anilludin1 moiety 1301 (FIG. 10) binds to a molecule to which the antibody1110, the growth factor 1120, the steroid 1140, the anti-angiogenicpeptide 1130, the integrin binding peptide 1150, the glycopeptide 1170,the lipid 1180, the peptide with biological activity 1190, or thecytokine 1195 binds and directs the illudin1 moiety 1301 to cellpopulations expressing the molecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via an azlactone linker 1230 (FIG. 10) to a pro-peptide 1160 iscleaved by an enzyme 1165 and thereafter binds to receptors for thepeptide and directs the illudin1 moiety 1301 to cell populationsexpressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with an azlactone linker 1230 (FIG. 10)to an illudin2 moiety 1302 binds to a molecule to which the antibody1110, the growth factor 1120, the steroid 1140, the anti-angiogenicpeptide 1130, the integrin binding peptide 1150, the glycopeptide 1170,the lipid 1180, the peptide with biological activity 1190, or thecytokine 1195 binds and directs the illudin2 moiety 1302 to cellpopulations expressing the molecule.

In an embodiment of the present invention, an illudin1 moiety 1301linked via an azlactone linker 1230 (FIG. 10) to a pro-peptide 1160 iscleaved by an enzyme 1165 and thereafter binds to receptors for thepeptide and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with an azlactone linker 1230 (FIG. 10)to a protein toxin 1330 binds to a molecule to which the antibody 1110,the growth factor 1120, the steroid 1140, the anti-angiogenic peptide1130, the integrin binding peptide 1150, the glycopeptide 1170, thelipid 1180, the peptide with biological activity 1190, or the cytokine1195 binds and directs the protein toxin 1330 to cell populationsexpressing the molecule.

In an embodiment of the present invention, a protein toxin 1330 linkedvia an azlactone linker 1230 (FIG. 10) to a pro-peptide 1160 is cleavedby an enzyme 1165 and thereafter binds to receptors for the peptide anddirects the protein toxin 1330 to cell populations expressing thereceptor.

In an embodiment of the present invention, a species selected from thegroup consisting of an antibody 1110, a growth factor 1120, a steroid1140, an anti-angiogenic peptide 1130, an integrin binding peptide 1150,a glycopeptide 1170, and a lipid 1180, a peptide with biologicalactivity 1190, a cytokine 1195 with an azlactone linker 1230 (FIG. 10)to a medicant 1350 binds to a molecule to which the antibody 1110, thegrowth factor 1120, the steroid 1140, the anti-angiogenic peptide 1130,the integrin binding peptide 1150, the glycopeptide 1170, the lipid1180, the peptide with biological activity 1190, or the cytokine 1195binds and directs the medicant 1350 to cell populations expressing themolecule.

In an embodiment of the present invention, a medicant 1350 linked via anazlactone linker 1230 (FIG. 10) to a pro-peptide 1160 is cleaved by anenzyme 1165 and thereafter binds to receptors for the peptide anddirects the medicant 1350 to cell populations expressing the receptor.

In an embodiment of the present invention, an anticancer medicant cantarget cancer cells with reduced toxicity to normal cells. In anembodiment of the present invention, an antibody linked to anacylfulvene can treat cell populations expressing the receptors. In anembodiment of the present invention, these compounds are useful intreatment of tumors in which the receptor is over expressed. In anembodiment of the present invention, these compounds are useful intreatment of cells in which the receptor acts as a marker and a target.In various embodiments of the present invention, pharmaceuticalcompositions comprising these compounds are used in the treatment oftumors in which the receptor is involved. In various embodiments of thepresent invention, methods of using the pharmaceutical compositionscomprise these compounds to treat tumors in which the receptor isinvolved. In various embodiments of the present invention,pharmaceutical compositions comprising these compounds are used in thetreatment of one or more of the following diseases: cancer,adenocarcinoma, carcinoma, breast cancer, prostate cancer, ovariancancer, endometrial cancer, neuroendocrine tumors, infertility,polycystic ovary syndrome, endometriosis, and precocious puberty.

In various embodiment of the present invention, an antibody linked to anacylfulvene moiety acts as a ligand for an Epidermal Growth Factor (EGF)receptor (EGF-R) (SEQ. ID. 143) and directs the acylfulvene to cellpopulations expressing the EGF-R. These compounds are useful as a meansof treating cell populations expressing the EGF-R. In an embodiment ofthe present invention, these compounds are useful in treatment of tumorsin which the EGF-R is over expressed. In an embodiment of the presentinvention, these compounds are useful in treatment of cells in which theEGF-R acts as a marker. In various embodiment of the present invention,these compounds are useful in agricultural applications in which theEGF-R acts as a marker of cell populations involved in agriculturalproduction. In various embodiment of the present invention, thesecompounds are useful in veterinary medicine in which the EGF-R acts as amarker of cell populations involved in pet reproduction. In variousembodiments of the present invention, pharmaceutical compositionscomprising these compounds are used in the treatment of tumors in whichthe EGF-R is involved. In various embodiments of the present invention,methods of using the pharmaceutical compositions comprise thesecompounds to treat tumors in which the GH-R is involved.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention is described with respect to specific embodimentsthereof. Additional features can be appreciated from the Figures inwhich:

FIG. 1 shows a schematic description of the Affinity Medicant Conjugate(AMC) 1000, where an Affinity Moiety (AM) 1100 is bound to a medicantmoiety (MM) 1300 via a Linker Unit (LU) 1200, according to variousembodiments of the invention;

FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M showthe structures of irofulvene medicant moieties, according to variousembodiments of the invention; FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG.2J, FIG. 2K, FIG. 2N and FIG. 2O show the structures of illudin medicantmoieties, according to various embodiments of the invention; FIG. 2Pshows the structure of azlactone acyfulvene medicant moiety (where R₂═H,CH₃, CH₂OH), according to an embodiment of the invention; FIG. 2Q showsthe structure of azlactone secondary hydroxyl illudin2 linkage medicantmoiety (where R₂═H, CH₃, CH₂OH), according to an embodiment of theinvention; FIG. 2R shows the structure of azlactone primary hydroxyllinkage illudin2 medicant moiety (where R₂═H, CH₃, CH₂OH), according toan embodiment of the invention; FIG. 2S and FIG. 2T show the structuresof the maleimide acylfulvene and maleimide illudin medicant moieties,respectively; FIG. 2U shows the structure of the maleic acylfulvenemedicant moiety, according to an embodiment of the invention; FIG. 2Vshows the structure of the maleic illudin medicant moiety, according toan embodiment of the invention;

FIG. 3A shows a schematic descriptions of an AMC 1001 where an Antibody(Ab) 1110 bound to a LU 1200 is bound to a medicant moiety (MM) 1300,according to various embodiments of the invention; FIG. 3B shows aschematic descriptions of an AMC 1002 where a Growth factor (Gf) 1120bound to a LU 1200 is bound to a medicant moiety (MM) 1300, according tovarious embodiments of the invention;

FIG. 4 shows the selective toxicity of an acylfulvene analog, accordingto various embodiments of the invention;

FIG. 5 shows the unique deoxynucleic acid (DNA) damage profile of anacylfulvene analog, according to an embodiment of the invention;

FIG. 6 shows the tumor regression of an acylfulvene analog, according toan embodiment of the invention;

FIG. 7 shows the multidrug resistance studies of an acylfulvene analog,according to an embodiment of the invention;

FIG. 8A shows the structure of the analog 211 (FIG. 20IB) attached viathe amino group using the sulfosuccinimidyl6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate (SMPT)linking reagent according to an embodiment of the invention; FIG. 8Bshows the structure of the analog 211 (FIG. 20IB) attached via the aminogroup using the sulfosuccimidyl4-(N-maleimido-methyl)cyclohexane-1-carboxylate (SMCC) linking reagentaccording to an embodiment of the invention; FIG. 8C shows the structureof the analog 211 (FIG. 20IB) attached via the amino group using thesulfosuccimidyl(4-iodo-acetyl)aminobenzoate (SIAB) linking reagentaccording to an embodiment of the invention;

FIG. 9A shows the structure of the analog 038 (FIG. 20BL) attached viathe carboxyl group using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) linking reagent according to an embodiment of theinvention; FIG. 9B shows the structure of the analog 038 (FIG. 20BL)attached via the carboxyl group using the1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide (CMC) linking reagentaccording to an embodiment of the invention; FIG. 9C shows the structureof the analog 038 (FIG. 20BL) attached via the carboxyl group using theN,N′-dicyclohexylcarbodiimide (DCC) linking reagent according to anembodiment of the invention;

FIG. 10A shows the structure of the analog 038 (FIG. 20BL) attached viathe carboxyl group using DCC or N,N′-diisopropylcarbodiimide (DIC)linking reagents in the presence of glycine according to variousembodiments of the invention; FIG. 10B shows the structure of the analog038 (FIG. 20BL) attached via the carboxyl group using DCC or DIC linkingreagents in the presence of alanine according to various embodiments ofthe invention; FIG. 10C shows the structure of the analog 106 (FIG.20EB) attached via the carboxyl group using DCC or DIC linking reagentsin the presence of valine according to various embodiments of theinvention;

FIG. 11A shows the structure of the analog 124 (FIG. 20ET) attached viathe carbonyl group using the 2-acetamido-4-mercaptobutyric acidhydrazide (AMBH) linking reagent according to an embodiment of theinvention; FIG. 11B shows the structure of the analog 124 (FIG. 20ET)attached via the carbonyl group using the p-azidobenzoyl hydrazide (ABH)linking reagent according to an embodiment of the invention; FIG. 11Cshows the structure of the analog 201 (FIG. 20HR) attached via the4-(N-maleimidomethyl) cyclohexanee-1-1carboxyl-hydrazide (M₂C₂H) linkingreagent according to an embodiment of the invention;

FIG. 12A shows the structure of the analog 010 (FIG. 20AJ) attached viathe aldehyde group using the 3-(2-pyridyldithio) propionyl hydrazide(PDPH) linking reagent according to an embodiment of the invention; FIG.12B shows the structure of the analog 010 (FIG. 20AJ) attached via thealdehyde group using the ABH linking reagent according to an embodimentof the invention; FIG. 12C shows the structure of the analog 011 (FIG.20AK) attached via 4-(4-N-maleimidophenyl)-butyric acid hydrazide (MPBH)linking reagent according to an embodiment of the invention;

FIG. 13A shows the structure of the analog 009 (FIG. 20AI) attached viathe alcohol group using the N,N′-carbonyldiimidazole (CDI) linkingreagent according to an embodiment of the invention; FIG. 13B shows thestructure of the analog 009 (FIG. 20AI) attached via the alcohol groupusing the N-hydroxysuccinimidyl chloroformate (HSC) linking reagentaccording to an embodiment of the invention; FIG. 13C shows thestructure of the medicant moiety Illudin M (FIG. 16A) attached via theN,N′-disuccinimidyl carbonate (DSC) linking reagent according to anembodiment of the invention;

FIG. 14A shows the structure of the analog 051 (FIG. 20BY) attached viathe sulfhydryl group using SMCC linking reagent according to anembodiment of the invention; FIG. 14B shows the structure of the analog051 (FIG. 20BY) attached via the sulfhydryl group using MPBH linkingreagent according to an embodiment of the invention; FIG. 14C shows thestructure of the analog 051 (FIG. 20BY) attached via the sulfhydrylgroup using PDPH linking reagent according to an embodiment of theinvention;

FIG. 15A shows the structures of 4-fluorosulfonyl benzoyl; FIG. 15Bshows the structure of 4-fluorosulfonyl benzoyl, 3-fluorosulfonylbenzoyl; FIG. 15C show the structures of 2-fluorosulfonyl benzoyl whereR₁, R₂, R₃, and R₄ independently denote H, F, Cl, Br and I;

FIG. 16A shows the structure of Illudin M; FIG. 16B shows amono-substituted product formed by reacting Illudin M on the secondaryhydroxyl to form 4-FSB linking reagent according to an embodiment of theinvention; FIG. 16C shows a mono-substituted product formed by reactingIlludin M on the secondary hydroxyl to form 2-FSB linking reagentaccording to an embodiment of the invention; FIG. 16D a mono-substitutedproduct formed by reacting Illudin M on the secondary hydroxyl to form3-FSB linking reagent according to an embodiment of the invention;

FIG. 17A shows the structure of the Illudin S FSB mono-substituted onthe primary hydroxy reagent according to an embodiment of the invention;FIG. 17B shows the structure of the Illudin S FSB mono-substituted onthe secondary hydroxy reagent according to an embodiment of theinvention; FIG. 17C shows the structure of the Illudin S FSBdi-substituted reagent according to an embodiment of the invention;

FIG. 18 shows the response of an ADC made by combining analog 316 (FIG.20MC) with the T101 antibody according to an embodiment of theinvention, where the per cent of control is plotted versus theconcentration of Namalva, a negative control (i.e., a cell line notexpressing T101) where the IC₅₀>1000 ng/mL and CEM, a positive control(i.e., a cell line expressing T101) where the IC₅₀<5 ng/mL, after a four(4) hour exposure and then eighteen (18) hour recovery, where the toxinto antibody ratio is 5:1 (as determined using a radiolabelled toxin) andwhere the concentration is in Illudin equivalents (ng of Illudinattached to antibody per mL of cell culture media), which demonstratesthe ability of T101-316 ADC to kill cells expressing T101 antigen ontheir surface but not cells that fail to express the T101 antigen;

FIG. 19A shows the activity (per cent survival of an ADC prepared withthe Rituxin antibody alone is proportional to CD20 expression on B cells(where MV522 cells are CD20 negative, 8392 cells express low numbers ofCD20, Raji express medium numbers of CD20 and Ramos express high numbersof CD20 molecules), and where B cells are relatively resistant toIlludins and irofulvens (48 hr IC₅₀>7,000 nM); FIG. 19B shows theactivity of the ADC prepared with the Rituxin antibody alone comparedwith an ADC of Rituxin with analog 218 (FIG. 20II) on Ramos cells; FIG.19C shows the activity of the ADC prepared with the Rituxin antibodyalone compared with an ADC of Rituxin with analog 218 (FIG. 20II) onRaji cells; FIG. 19D shows the activity of the ADC prepared with theRituxin antibody alone compared with an ADC of Rituxin with analog 218(FIG. 20II) on 8392 cells; FIG. 19E shows the activity of the ADCprepared with the Rituxin antibody alone compared with an ADC of Rituxinwith analog 218 (FIG. 20II) on MV522 cells (where MV522 cells grew inthe presence of the Rituxan—analog 218 ADC (FIG. 20II) but at a slowerrate than in the absence of analog 218 ADC);

FIG. 20AA shows analog 001 according to an embodiment of the invention;FIG. 20AB shows analog 002 according to an embodiment of the invention;FIG. 20AC shows analog 003 according to an embodiment of the invention;FIG. 20AD shows analog 004 according to an embodiment of the invention;FIG. 20AE shows analog 005 according to an embodiment of the invention;FIG. 20AF shows analog 006 according to an embodiment of the invention;FIG. 20AG shows analog 007 according to an embodiment of the invention;FIG. 20AH shows analog 008 according to an embodiment of the invention;FIG. 20AI shows analog 009 according to an embodiment of the invention;FIG. 20AJ shows analog 010 according to an embodiment of the invention;FIG. 20AK shows analog 011 according to an embodiment of the invention;FIG. 20AL shows analog 012 according to an embodiment of the invention;FIG. 20AM shows analog 013 according to an embodiment of the invention;FIG. 20AN shows analog 014 according to an embodiment of the invention;FIG. 20AO shows analog 015 according to an embodiment of the invention;FIG. 20AP shows analog 016 according to an embodiment of the invention;FIG. 20AQ shows analog 017 according to an embodiment of the invention;FIG. 20AR shows analog 018 according to an embodiment of the invention;FIG. 20AS shows analog 019 according to an embodiment of the invention;FIG. 20AT shows analog 020 according to an embodiment of the invention;FIG. 20AU shows analog 021 according to an embodiment of the invention;FIG. 20AV shows analog 022 according to an embodiment of the invention;FIG. 20AW shows analog 023 according to an embodiment of the invention;FIG. 20AX shows analog 024 according to an embodiment of the invention;FIG. 20AY shows analog 025 according to an embodiment of the invention;FIG. 20AZ shows analog 026 according to an embodiment of the invention;FIG. 20BA shows analog 027 according to an embodiment of the invention;FIG. 20BB shows analog 028 according to an embodiment of the invention;FIG. 20BC shows analog 029 according to an embodiment of the invention;FIG. 20BD shows analog 030 according to an embodiment of the invention;FIG. 20BE shows analog 031 according to an embodiment of the invention;FIG. 20BF shows analog 032 according to an embodiment of the invention;FIG. 20BG shows analog 033 according to an embodiment of the invention;FIG. 20BH shows analog 034 according to an embodiment of the invention;FIG. 20BI shows analog 035 according to an embodiment of the invention;FIG. 20BJ shows analog 036 according to an embodiment of the invention;FIG. 20BK shows analog 037 according to an embodiment of the invention;FIG. 20BL shows analog 038 according to an embodiment of the invention;FIG. 20BM shows analog 039 according to an embodiment of the invention;FIG. 20BN shows analog 040 according to an embodiment of the invention;FIG. 20BO shows analog 041 according to an embodiment of the invention;FIG. 20BP shows analog 042 according to an embodiment of the invention;FIG. 20BQ shows analog 043 according to an embodiment of the invention;FIG. 20BR shows analog 044 according to an embodiment of the invention;FIG. 20BS shows analog 045 according to an embodiment of the invention;FIG. 20BT shows analog 046 according to an embodiment of the invention;FIG. 20BU shows analog 047 according to an embodiment of the invention;FIG. 20BV shows analog 048 according to an embodiment of the invention;FIG. 20BW shows analog 049 according to an embodiment of the invention;FIG. 20BX shows analog 050 according to an embodiment of the invention;FIG. 20BY shows analog 051 according to an embodiment of the invention;FIG. 20BZ shows analog 052 according to an embodiment of the invention;FIG. 20CA shows analog 053 according to an embodiment of the invention;FIG. 20CB shows analog 054 according to an embodiment of the invention;FIG. 20CC shows analog 055 according to an embodiment of the invention;FIG. 20CD shows analog 056 according to an embodiment of the invention;FIG. 20CE shows analog 057 according to an embodiment of the invention;FIG. 20CF shows analog 058 according to an embodiment of the invention;FIG. 20CG shows analog 059 according to an embodiment of the invention;FIG. 20CH shows analog 060 according to an embodiment of the invention;FIG. 20CI shows analog 061 according to an embodiment of the invention;FIG. 20CJ shows analog 062 according to an embodiment of the invention;FIG. 20CK shows analog 063 according to an embodiment of the invention;FIG. 20CL shows analog 064 according to an embodiment of the invention;FIG. 20CM shows analog 065 according to an embodiment of the invention;FIG. 20CN shows analog 066 according to an embodiment of the invention;FIG. 20CO shows analog 067 according to an embodiment of the invention;FIG. 20CP shows analog 068 according to an embodiment of the invention;FIG. 20CQ shows analog 069 according to an embodiment of the invention;FIG. 20CR shows analog 070 according to an embodiment of the invention;FIG. 20CS shows analog 071 according to an embodiment of the invention;FIG. 20CT shows analog 072 according to an embodiment of the invention;FIG. 20CU shows analog 073 according to an embodiment of the invention;FIG. 20CV shows analog 074 according to an embodiment of the invention;FIG. 20CW shows analog 075 according to an embodiment of the invention;FIG. 20CX shows analog 076 according to an embodiment of the invention;FIG. 20CY shows analog 077 according to an embodiment of the invention;FIG. 20CZ shows analog 078 according to an embodiment of the invention;FIG. 20DA shows analog 079 according to an embodiment of the invention;FIG. 20DB shows analog 080 according to an embodiment of the invention;FIG. 20DC shows analog 081 according to an embodiment of the invention;FIG. 20DD shows analog 082 according to an embodiment of the invention;FIG. 20DE shows analog 083 according to an embodiment of the invention;FIG. 20DF shows analog 084 according to an embodiment of the invention;FIG. 20DG shows analog 085 according to an embodiment of the invention;FIG. 20DH shows analog 086 according to an embodiment of the invention;FIG. 20DI shows analog 087 according to an embodiment of the invention;FIG. 20DJ shows analog 088 according to an embodiment of the invention;FIG. 20DK shows analog 089 according to an embodiment of the invention;FIG. 20DL shows analog 090 according to an embodiment of the invention;FIG. 20DM shows analog 091 according to an embodiment of the invention;FIG. 20DN shows analog 092 according to an embodiment of the invention;FIG. 20DO shows analog 093 according to an embodiment of the invention;FIG. 20DP shows analog 094 according to an embodiment of the invention;FIG. 20DQ shows analog 095 according to an embodiment of the invention;FIG. 20DR shows analog 096 according to an embodiment of the invention;FIG. 20DS shows analog 097 according to an embodiment of the invention;FIG. 20DT shows analog 098 according to an embodiment of the invention;FIG. 20DU shows analog 099 according to an embodiment of the invention;FIG. 20DV shows analog 100 according to an embodiment of the invention;FIG. 20DW shows analog 101 according to an embodiment of the invention;FIG. 20DX shows analog 102 according to an embodiment of the invention;FIG. 20DY shows analog 103 according to an embodiment of the invention;FIG. 20DZ shows analog 104 according to an embodiment of the invention;FIG. 20EA shows analog 105 according to an embodiment of the invention;FIG. 20EB shows analog 106 according to an embodiment of the invention;FIG. 20EC shows analog 107 according to an embodiment of the invention;FIG. 20ED shows analog 108 according to an embodiment of the invention;FIG. 20EE shows analog 109 according to an embodiment of the invention;FIG. 20EF shows analog 110 according to an embodiment of the invention;FIG. 20EG shows analog 111 according to an embodiment of the invention;FIG. 20EH shows analog 112 according to an embodiment of the invention;FIG. 20EI shows analog 113 according to an embodiment of the invention;FIG. 20EJ shows analog 114 according to an embodiment of the invention;FIG. 20EK shows analog 115 according to an embodiment of the invention;FIG. 20EL shows analog 116 according to an embodiment of the invention;FIG. 20EM shows analog 117 according to an embodiment of the invention;FIG. 20EN shows analog 118 according to an embodiment of the invention;FIG. 20EO shows analog 119 according to an embodiment of the invention;FIG. 20EP shows analog 120 according to an embodiment of the invention;FIG. 20EQ shows analog 121 according to an embodiment of the invention;FIG. 20ER shows analog 122 according to an embodiment of the invention;FIG. 20ES shows analog 123 according to an embodiment of the invention;FIG. 20ET shows analog 124 according to an embodiment of the invention;FIG. 20EU shows analog 125 according to an embodiment of the invention;FIG. 20EV shows analog 126 according to an embodiment of the invention;FIG. 20EW shows analog 127 according to an embodiment of the invention;FIG. 20EX shows analog 128 according to an embodiment of the invention;FIG. 20EY shows analog 129 according to an embodiment of the invention;FIG. 20EZ shows analog 130 according to an embodiment of the invention;FIG. 20FA shows analog 131 according to an embodiment of the invention;FIG. 20FB shows analog 133 according to an embodiment of the invention;FIG. 20FC shows analog 134 according to an embodiment of the invention;FIG. 20FD shows analog 135 according to an embodiment of the invention;FIG. 20FE shows analog 136 according to an embodiment of the invention;FIG. 20FF shows analog 137 according to an embodiment of the invention;FIG. 20FG shows analog 138 according to an embodiment of the invention;FIG. 20FH shows analog 139 according to an embodiment of the invention;FIG. 20FI shows analog 140 according to an embodiment of the invention;FIG. 20FJ shows analog 141 according to an embodiment of the invention;FIG. 20FK shows analog 142 according to an embodiment of the invention;FIG. 20FL shows analog 143 according to an embodiment of the invention;FIG. 20FM shows analog 144 according to an embodiment of the invention;FIG. 20FN shows analog 145 according to an embodiment of the invention;FIG. 20FO shows analog 146 according to an embodiment of the invention;FIG. 20FP shows analog 147 according to an embodiment of the invention;FIG. 20FQ shows analog 148 according to an embodiment of the invention;FIG. 20FR shows analog 149 according to an embodiment of the invention;FIG. 20FS shows analog 150 according to an embodiment of the invention;FIG. 20FT shows analog 151 according to an embodiment of the invention;FIG. 20FU shows analog 152 according to an embodiment of the invention;FIG. 20FV shows analog 153 according to an embodiment of the invention;FIG. 20FW shows analog 154 according to an embodiment of the invention;FIG. 20FX shows analog 155 according to an embodiment of the invention;FIG. 20FY shows analog 156 according to an embodiment of the invention;FIG. 20FZ shows analog 157 according to an embodiment of the invention;FIG. 20GA shows analog 158 according to an embodiment of the invention;FIG. 20 GB shows analog 159 according to an embodiment of the invention;FIG. 20GC shows analog 160 according to an embodiment of the invention;FIG. 20GD shows analog 161 according to an embodiment of the invention;FIG. 20GE shows analog 162 according to an embodiment of the invention;FIG. 20GF shows analog 163 according to an embodiment of the invention;FIG. 20GG shows analog 164 according to an embodiment of the invention;FIG. 20GH shows analog 165 according to an embodiment of the invention;FIG. 20GI shows analog 166 according to an embodiment of the invention;FIG. 20GJ shows analog 167 according to an embodiment of the invention;FIG. 20GK shows analog 168 according to an embodiment of the invention;FIG. 20GL shows analog 169 according to an embodiment of the invention;FIG. 20GM shows analog 170 according to an embodiment of the invention;FIG. 20GN shows analog 171 according to an embodiment of the invention;FIG. 20GO shows analog 172 according to an embodiment of the invention;FIG. 20GP shows analog 173 according to an embodiment of the invention;FIG. 20GQ shows analog 174 according to an embodiment of the invention;FIG. 20GR shows analog 175 according to an embodiment of the invention;FIG. 20GS shows analog 176 according to an embodiment of the invention;FIG. 20GT shows analog 177 according to an embodiment of the invention;FIG. 20GU shows analog 178 according to an embodiment of the invention;FIG. 20GV shows analog 179 according to an embodiment of the invention;FIG. 20GW shows analog 180 according to an embodiment of the invention;FIG. 20GX shows analog 181 according to an embodiment of the invention;FIG. 20GY shows analog 182 according to an embodiment of the invention;FIG. 20GZ shows analog 183 according to an embodiment of the invention;FIG. 20HA shows analog 184 according to an embodiment of the invention;FIG. 20HB shows analog 185 according to an embodiment of the invention;FIG. 20HC shows analog 186 according to an embodiment of the invention;FIG. 20HD shows analog 187 according to an embodiment of the invention;FIG. 20HE shows analog 188 according to an embodiment of the invention;FIG. 20HF shows analog 189 according to an embodiment of the invention;FIG. 20HG shows analog 190 according to an embodiment of the invention;FIG. 20HH shows analog 191 according to an embodiment of the invention;FIG. 20HI shows analog 192 according to an embodiment of the invention;FIG. 20HJ shows analog 193 according to an embodiment of the invention;FIG. 20HK shows analog 194 according to an embodiment of the invention;FIG. 20HL shows analog 195 according to an embodiment of the invention;FIG. 20HM shows analog 196 according to an embodiment of the invention;FIG. 20HN shows analog 197 according to an embodiment of the invention;FIG. 20HO shows analog 198 according to an embodiment of the invention;FIG. 20HP shows analog 199 according to an embodiment of the invention;FIG. 20HQ shows analog 200 according to an embodiment of the invention;

FIG. 20HR shows analog 201 according to an embodiment of the invention;FIG. 20HS shows analog 202 according to an embodiment of the invention;FIG. 20HT shows analog 203 according to an embodiment of the invention;FIG. 20HU shows analog 204 according to an embodiment of the invention;FIG. 20HV shows analog 205 according to an embodiment of the invention;FIG. 20HW shows analog 206 according to an embodiment of the invention;FIG. 20HX shows analog 207 according to an embodiment of the invention;FIG. 20HY shows analog 208 according to an embodiment of the invention;FIG. 20HZ shows analog 209 according to an embodiment of the invention;FIG. 20IA shows analog 210 according to an embodiment of the invention;FIG. 20IB shows analog 211 according to an embodiment of the invention;FIG. 20IC shows analog 212 according to an embodiment of the invention;FIG. 201D shows analog 213 according to an embodiment of the invention;FIG. 20IE shows analog 214 according to an embodiment of the invention;FIG. 20IF shows analog 215 according to an embodiment of the invention;FIG. 20IG shows analog 216 according to an embodiment of the invention;FIG. 20IH shows analog 217 according to an embodiment of the invention;FIG. 20II shows analog 218 according to an embodiment of the invention;FIG. 20IJ shows analog 219 according to an embodiment of the invention;FIG. 20IK shows analog 220 according to an embodiment of the invention;FIG. 20IL shows analog 221 according to an embodiment of the invention;FIG. 20IM shows analog 222 according to an embodiment of the invention;FIG. 20IN shows analog 223 according to an embodiment of the invention;FIG. 20IO shows analog 224 according to an embodiment of the invention;FIG. 20IP shows analog 225 according to an embodiment of the invention;FIG. 20IQ shows analog 226 according to an embodiment of the invention;FIG. 20IR shows analog 227 according to an embodiment of the invention;FIG. 20IS shows analog 228 according to an embodiment of the invention;FIG. 20IT shows analog 229 according to an embodiment of the invention;FIG. 201U shows analog 230 according to an embodiment of the invention;FIG. 20IV shows analog 231 according to an embodiment of the invention;FIG. 20IW shows analog 232 according to an embodiment of the invention;FIG. 20IX shows analog 233 according to an embodiment of the invention;FIG. 20IY shows analog 234 according to an embodiment of the invention;FIG. 20IZ shows analog 235 according to an embodiment of the invention;FIG. 20JA shows analog 236 according to an embodiment of the invention;FIG. 20JB shows analog 237 according to an embodiment of the invention;FIG. 20JC shows analog 238 according to an embodiment of the invention;FIG. 20JD shows analog 239 according to an embodiment of the invention;FIG. 20JE shows analog 240 according to an embodiment of the invention;FIG. 20JF shows analog 241 according to an embodiment of the invention;FIG. 20JG shows analog 242 according to an embodiment of the invention;FIG. 20JH shows analog 243 according to an embodiment of the invention;FIG. 20JI shows analog 244 according to an embodiment of the invention;FIG. 20JJ shows analog 245 according to an embodiment of the invention;FIG. 20JK shows analog 246 according to an embodiment of the invention;FIG. 20JL shows analog 247 according to an embodiment of the invention;FIG. 20JM shows analog 248 according to an embodiment of the invention;FIG. 20JN shows analog 249 according to an embodiment of the invention;FIG. 20JO shows analog 250 according to an embodiment of the invention;FIG. 20JP shows analog 251 according to an embodiment of the invention;FIG. 20JQ shows analog 252 according to an embodiment of the invention;FIG. 20JR shows analog 253 according to an embodiment of the invention;FIG. 20JS shows analog 254 according to an embodiment of the invention;FIG. 20JT shows analog 255 according to an embodiment of the invention;FIG. 20JU shows analog 256 according to an embodiment of the invention;FIG. 20JV shows analog 257 according to an embodiment of the invention;FIG. 20JW shows analog 258 according to an embodiment of the invention;FIG. 20JX shows analog 259 according to an embodiment of the invention;FIG. 20JY shows analog 260 according to an embodiment of the invention;FIG. 20JZ shows analog 261 according to an embodiment of the invention;FIG. 20KA shows analog 262 according to an embodiment of the invention;FIG. 20KB shows analog 263 according to an embodiment of the invention;FIG. 20KC shows analog 264 according to an embodiment of the invention;FIG. 20KD shows analog 265 according to an embodiment of the invention;FIG. 20KE shows analog 266 according to an embodiment of the invention;FIG. 20KF shows analog 267 according to an embodiment of the invention;FIG. 20KG shows analog 268 according to an embodiment of the invention;FIG. 20KH shows analog 269 according to an embodiment of the invention;FIG. 20KI shows analog 270 according to an embodiment of the invention;FIG. 20KJ shows analog 271 according to an embodiment of the invention;FIG. 20KK shows analog 272 according to an embodiment of the invention;FIG. 20KL shows analog 273 according to an embodiment of the invention;FIG. 20KM shows analog 274 according to an embodiment of the invention;FIG. 20KN shows analog 275 according to an embodiment of the invention;FIG. 20KO shows analog 276 according to an embodiment of the invention;FIG. 20KP shows analog 277 according to an embodiment of the invention;FIG. 20KQ shows analog 278 according to an embodiment of the invention;FIG. 20KR shows analog 279 according to an embodiment of the invention;FIG. 20KS shows analog 280 according to an embodiment of the invention;FIG. 20KT shows analog 281 according to an embodiment of the invention;FIG. 20KU shows analog 282 according to an embodiment of the invention;FIG. 20KV shows analog 283 according to an embodiment of the invention;FIG. 20KW shows analog 284 according to an embodiment of the invention;FIG. 20KX shows analog 285 according to an embodiment of the invention;FIG. 20KY shows analog 286 according to an embodiment of the invention;FIG. 20KZ shows analog 287 according to an embodiment of the invention;FIG. 20LA shows analog 288 according to an embodiment of the invention;FIG. 20LB shows analog 289 according to an embodiment of the invention;FIG. 20LC shows analog 290 according to an embodiment of the invention;FIG. 20LD shows analog 291 according to an embodiment of the invention;FIG. 20LE shows analog 292 according to an embodiment of the invention;FIG. 20LF shows analog 293 according to an embodiment of the invention;FIG. 20LG shows analog 294 according to an embodiment of the invention;FIG. 20LH shows analog 295 according to an embodiment of the invention;FIG. 20LI shows analog 296 according to an embodiment of the invention;FIG. 20LJ shows analog 297 according to an embodiment of the invention;FIG. 20LK shows analog 298 according to an embodiment of the invention;FIG. 20LL shows analog 299 according to an embodiment of the invention;FIG. 20LM shows analog 300 according to an embodiment of the invention;FIG. 20LN shows analog 301 according to an embodiment of the invention;FIG. 20LO shows analog 302 according to an embodiment of the invention;FIG. 20LP shows analog 303 according to an embodiment of the invention;FIG. 20LQ shows analog 304 according to an embodiment of the invention;FIG. 20LR shows analog 305 according to an embodiment of the invention;FIG. 20LS shows analog 306 according to an embodiment of the invention;FIG. 20LT shows analog 307 according to an embodiment of the invention;FIG. 20LU shows analog 308 according to an embodiment of the invention;FIG. 20LV shows analog 309 according to an embodiment of the invention;FIG. 20LW shows analog 310 according to an embodiment of the invention;FIG. 20LX shows analog 311 according to an embodiment of the invention;FIG. 20LY shows analog 312 according to an embodiment of the invention;FIG. 20LZ shows analog 313 according to an embodiment of the invention;FIG. 20MA shows analog 314 according to an embodiment of the invention;FIG. 20MB shows analog 315 according to an embodiment of the invention;FIG. 20MC shows analog 316 according to an embodiment of the invention;

FIG. 21A shows analog 20 (FIG. 20AT) linked to DSP according to anembodiment of the invention; FIG. 21B shows analog 20 (FIG. 20AT) linkedto DTME according to an embodiment of the invention; FIG. 21C showsanalog 20 (FIG. 20AT) linked to SMPT according to an embodiment of theinvention;

FIG. 22A shows an illudin analog according to various embodiments of theinvention; FIG. 22B shows a syn-illudin analog according to variousembodiments of the invention; and

FIG. 22C and FIG. 22D show acylfulvene analogs according to variousembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The transitional term “comprising” is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, un-recited elements or method steps.

The transitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim, but does not exclude additionalcomponents or steps that are unrelated to the invention such asimpurities ordinarily associated with a composition.

The transitional phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

As used herein, the term “receptor for a biologically activepolypeptide” means a receptor which can bind a biologically activepeptide conjugate.

As used herein, the term “cell population” is used to describe a set orsubset of cells expressing a receptor.

As used herein, the terms “analog”, “medicant” and “medicant moiety” areused interchangeably and comprise synthetic and naturally occurringdrugs, toxins, nutraceuticals and other cytoactive, anti-inflammatoryand bioactive molecules including Doxorubicin (Immunomedics),auristatins E (Seattle Genetics), auristatins F (Celdex), monomethylauristatin E (MMAE) (Amgen), monomethyl auristatin F (MMAF) (Astelles),maytanasines (Immunogen), DM1 (Biotest), DM4 (Amgen), calicheamicin(CellTech), irinotecan, folate, SN38 (Immunomedics),Pyrrolobenzodiazepines (Seattle Genetics), MGBA a duocarmycin derivative(Medarex), thalidomides, taxanes, penicillins, Trastuzumab emtansine(Genentech for Breast cancer uses maytanasine derive DM-1). A medicantincludes analogs 192 (FIG. 20IH), 197 (FIG. 20HN), 272 (FIG. 20KK), 273(FIG. 20KL), 274 (FIG. 20KM), 290 (FIG. 20LC), 291 (FIG. 20LD), 292(FIG. 20LE), 293 (FIG. 20LF) (i.e., acylfulvenes linked to thalidomide,cephalosporin or colchicines derivatives). Some of the above analogs arestand alone drugs, but can be used as a medicant moiety in an affinitydrug conjugate according to various embodiments of the invention.

As used herein, the phrase “peptide receptor” includes peptide hormonereceptors, protein hormone receptors, chemotactic receptors andchemokine receptors.

As used herein, the term “receptor” includes growth factor receptors,peptide hormone receptors, peptide receptors, steroid hormone receptors,steroid receptors and lipid receptors.

As used herein, phrase “affinity medicant conjugate” is an AffinityMoiety covalently bound to a medicant moiety, and includes antibodymedicant conjugates, where the antibody is directed to a specificreceptor. As used herein the phrase ‘Affinity Moiety’ includesantibodies, antibody fragments, peptides, proteins, growth factors,steroids, and lipids, where the antibodies, antibody fragments,peptides, proteins, growth factors, steroids, folate or lipids have anaffinity for a specific receptor, receptors, is processed by an enzymeto produce a ligand that has an affinity for a specific receptor orotherwise directs the Affinity Moiety to a specific subset of cells. A‘medicant moiety’ includes a group bound to an Affinity Moiety, whichwhen released acts as a medicant.

As used herein, the term “Affinity Moiety” (AM) is used to describe achemical group or molecule that can bind a receptor or proteins. An AMis understood to have a minimum binding affinity greater thanapproximately 1×10⁻³ M affinity. As used herein, the term AM includes“ligands”, “ligand moieties”, “affinity unit” and an AM modified toinclude a linker. As used herein, the phrase “an affinity moietydirected to a peptide receptor” is used to describe a molecule or aportion of a molecule which has a binding affinity to the peptidereceptor greater than approximately 1×10⁻¹⁰ M. In this rangeapproximately means 1×10⁻⁹ M to 1×10⁻¹¹ M. In an embodiment of theinvention, an AM directed to a peptide receptor has a binding affinityto the peptide receptor greater than approximately 1×10⁻¹² M. In thisrange approximately means 1×10⁻¹¹ M to 1×10⁻¹³ M.

As used herein, the term “cytoactive” (which is abbreviated as “CA”) isused to describe a small molecule that disrupts a cellular process,modulates a cellular process or otherwise affects the normal function ofthe cell. As used herein, the term “toxin” or “toxic” is used todescribe a small molecule which interferes with RNA or DNA synthesis,causes RNA or DNA strand scission, blocks cell cycling, division,replication or is otherwise cytotoxic to the cell. As used herein, theterm “toxin moiety” is used to describe a toxin modified to include alinker. As used herein, the phrase “a moiety possessing cellcytotoxicity” is used to describe a toxin moiety which when given in theconcentration range of approximately 1×10⁻³ M to approximately 1×10⁻⁹Mresults in inhibition of DNA synthesis or proliferation in anappropriate cultured cell line and/or when administered intravenously toan animal in the dosage approximately 1×10⁻⁶ g to approximately 1×10⁻⁹ gof the compound per kilogram of body weight of the animal results in invivo cell death. As used herein, the term “ablated” is used to describea reduction in the cell population of between approximately 50% andapproximately 95%. In this range approximately means plus or minus five(5) per cent. In an embodiment of the invention, a toxin moiety ablatinga cell population reduces the cell population by approximately 100 percent. As used herein, the term “impaired” is used to describe areduction in the cell population of between approximately 30% andapproximately 50%. In this range approximately means plus or minus ten(10) per cent.

As used herein, the term “linker” is used to describe one or morecovalently bonded groups of atoms that are covalently bonded to amedicant moiety and an AM. For example a linker can be covalently boundto both an acylfulvene moiety and to an antibody or other ligand moietywith an affinity for a receptor.

As used herein, the term “non releasable linker” is used to describe alinker covalently bound to an AM and a medicant moiety in which the AMand the medicant moiety remain covalently bound to the linker afterinternalization and exposure to both reducing and acidic environments ofvesicles within the cell. As used herein, the term “membranepermeability” is used to describe a compound comprising a linkercovalently bound to an AM and an acylfulvene moiety, where the compoundcan diffuse across membranes within the cell.

As used herein, the term “transmembrane receptor” means a protein thatspans the plasma membrane of a cell with the extracellular domain of theprotein having the ability to bind an AM and the intracellular domainhaving an activity such as activation of G protein signaling which isinduced upon the AM binding.

As used herein, the term “seven transmembrane receptor” is atransmembrane receptor including a transmembrane domain where theprotein spans the cell membrane in seven (7) regions.

As used herein, the term “G-protein coupled receptor” means a seventransmembrane domain receptor which transduces a biological signal viaG-protein coupling.

As used herein, the term “conjugated” or “conjugate” means a chemicalcompound that is formed by joining two or more compounds with one ormore chemical bonds or linkers. In an embodiment of the invention, anantibody and a medicant form a conjugate.

As used herein, the term “antibody” herein is used in the broadest senseand specifically covers intact antibodies, monoclonal antibodies,polyclonal antibodies, mono-specific antibodies, multi-specificantibodies (e.g., bi-specific antibodies), and antibody fragments thatexhibit the desired biological activity, including those antibodiesdirected against Alk, Alk fusion proteins, CD 2 (SEQ. ID. 001),CD3epsilon (SEQ. ID. 002), CD5 (SEQ. ID. 003), CD7 (SEQ. ID. 004), CD19(SEQ. ID. 005), CD20 (SEQ. ID. 006), CD22 (SEQ. ID. 007), CD25 (SEQ. ID.008), CD30 (SEQ. ID. 009), CD33 (SEQ. ID. 010), CD37 (SEQ. ID. 011),CD44 (SEQ. ID. 012), CD44v6 (SEQ. ID. 013), CD56 (SEQ. ID. 014), CD70(SEQ. ID. 015), CD74 (SEQ. ID. 016), CD79 (SEQ. ID. 017), CD79b (SEQ.ID. 018), CD 80 (SEQ. ID. 019), CD 86 (SEQ. ID. 020), CD138 (syndecan 1)(SEQ. ID. 021), CAIX (SEQ. ID. 022), Integrin alphaVbeta 3 (SEQ. ID.023), EphA2 (SEQ. ID. 024), Cryptol (SEQ. ID. 025), CanAg (SEQ. ID.026), ENPP3 (SEQ. ID. 027), Nectin-4 (SEQ. ID. 028), Mesothelin (SEQ.ID. 029), Lewis Y (SEQ. ID. 030), EGFRvIII (SEQ. ID. 031), SLC44A4 (SEQ.ID. 032), EBTR (endothelin) (SEQ. ID. 033), erbB2/neu/HER2 (SEQ. ID.034), Transferrin receptor (SEQ. ID. 035), 55 kDa breast cancer antigen,72 kDa TAA, GPNMB (osteoactivin) (SEQ. ID. 038), CA-IX (SEQ. ID. 039),CEA (CD66e) (SEQ. ID. 040), CEACAMS (SEQ. ID. 041), PSMA (SEQ. ID. 042),CAl25 (MUC16) (SEQ. ID. 043), Mud 1 (CA6) (SEQ. ID. 044), Melanomaglycoprotein NMB (SEQ. ID. 045), IL-2R (SEQ. ID. 046), IL13R (SEQ. ID.047), TACSTD2 (TROP2 or EGP1) (SEQ. ID. 048), Folate receptor 1 (SEQ.ID. 049), Mucin 16 (SEQ. ID. 050), Endothelin receptor ETB (SEQ. ID.051), STEAP1 (SEQ. ID. 052), SLC44A4 (AGS-5) (SEQ. ID. 053), AGS-16(SEQ. ID. 054), and Guanylyl cyclase C (SEQ. ID. 055). An intactantibody has primarily two regions: a variable region and a constantregion. The variable region binds to and interacts with a targetantigen. The variable region includes a complementary determinusngregion (CDR) that recognizes and binds to a specific binding site on aparticular antigen. The constant region may be recognized by andinteract with the immune system. An antibody can be of any type or class(e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2). The antibody can be derived from any suitablespecies. In some embodiments, the antibody is of human or murine origin.An antibody can be, for example, human, humanized or chimeric.

As used herein, the terms “specifically binds” and “specific binding”refer to antibody binding to a predetermined antigen. Typically, theantibody binds with an affinity of at least about 1×10⁷ M, and binds tothe predetermined antigen with an affinity that is at least two-foldgreater than its affinity for binding to a non-specific antigen (e.g.,Bovine Serum Albumin, casein) other than the predetermined antigen or aclosely-related antigen.

As used herein, the term “monoclonal antibody” as used herein refers toan antibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts and includes “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical to or homologouswith the corresponding sequence of antibodies derived from a particularspecies or belonging to a particular antibody class or subclass, whilethe remainder of the chain(s) is identical to or homologous with thecorresponding sequences of antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biologicalactivity. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method.

As used herein, an “intact antibody” is one which comprises anantigen-binding variable region as well as a light chain constant domain(C_(L)) and heavy chain constant domains, C_(H1), C_(H2), C_(H3) andC_(H4), as appropriate for the antibody class. The constant domains maybe native sequence constant domains (e.g., human native sequenceconstant domains) or amino acid sequence variants thereof.

As used herein, the term an “intact antibody” may have one or more“effector functions”, which refers to those biological activitiesattributable to the Fc region (e.g., a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include complement dependent cytotoxicity,antibody-dependent cell-mediated cytotoxicity (AMCC) andantibody-dependent cell-mediated phagocytosis.

As used herein, the term an “antibody fragment” comprises a portion ofan intact antibody, preferably comprising the antigen-binding orvariable region thereof. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments, di-abodies, tri-abodies, tetrabodies,linear antibodies, single-chain antibody molecules, scFv, scFv-Fc,multi-specific antibody fragments formed from antibody fragment(s), afragment(s) produced by a Fab expression library, or an epitope-bindingfragments of any of the above which immuno specifically bind to a targetantigen (e.g., a cancer cell antigen, a viral antigen or a microbialantigen).

As used herein, the term “variable” in the context of an antibody refersto certain portions of the variable domains of the antibody that differextensively in sequence and are used in the binding and specificity ofeach particular antibody for its particular antigen. This variability isconcentrated in three segments called “hypervariable regions” in thelight chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the Framework Regions(FRs). The variable domains of native heavy and light chains eachcomprise four FRs connected by three hypervariable regions.

As used herein, the term “hypervariable region” when used herein refersto the amino acid residues of an antibody which are responsible forantigen-binding. The hypervariable region generally comprises amino acidresidues from a “complementarity determining region” or “CDR” (e.g.,residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (L3) in the heavychain variable domain, and/or those residues from a “hypervariable loop”(e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (142) and 96-101 (H3) in the heavychain variable domain. FR residues are those variable domain residuesother than the hypervariable region residues as herein defined.

As used herein, a “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Typically, the Fvpolypeptide further comprises a polypeptide linker between the V_(H) andV_(L) domains which enables the scFv to form the desired structure forantigen binding.

As used herein, the term “di-abody” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a variableheavy domain (V_(H)) connected to a variable light domain (V_(L)) in thesame polypeptide chain. By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

As used herein, “Humanized” forms of non-human (e.g., rodent) antibodiesare chimeric antibodies that contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

As used herein, “isolated” means separated from other components of (a)a natural source, such as a plant or animal cell or cell culture, or (b)a synthetic organic chemical reaction mixture. As used herein,“purified” means that when isolated, the isolate contains at least 95%,and in another aspect at least 98%, of a compound (e.g., a conjugate) byweight of the isolate.

As used herein, an “isolated” antibody is one which has been identifiedand separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the antibodywill be purified (1) to greater than 95% by weight of antibody asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

As used herein, an antibody which “induces apoptosis” is one whichinduces programmed cell death as determined by binding of annexin V,fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum,cell fragmentation, and/or formation of membrane vesicles (calledapoptotic bodies). The cell is a tumor cell, e.g., a breast, ovarian,stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,pancreatic or bladder cell. Various methods are available for evaluatingthe cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNAfragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells.

As used herein, the term “therapeutically effective amount” refers to anamount of a medicant effective to treat a disease or disorder in amammal. In the case of cancer, the therapeutically effective amount ofthe medicant may reduce the number of cancer cells; reduce the tumorsize; inhibit (i.e., slow to some extent and preferably stop) cancercell infiltration into peripheral organs; inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; inhibit, to some extent,tumor growth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the medicant may inhibit thegrowth of and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. For cancer therapy, efficacy can, for example, be measured byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR).

As used herein, the term “substantial amount” refers to a majority, i.e.greater than approximately fifty per cent (50%) of a population, of amixture or a sample. In this range approximately means plus or minus tenper cent (10%).

As used herein, the term “intracellular metabolite” refers to a compoundresulting from a metabolic process or reaction inside a cell on anAffinity Medicant Linker conjugate (e.g., an Antibody Drug Conjugate(AMC)). The metabolic process or reaction may be an enzymatic processsuch as proteolytic cleavage of a peptide linker of the AMC.Intracellular metabolites include, but are not limited to, antibodiesand free medicant which have undergone intracellular cleavage afterentry, diffusion, uptake or transport into a cell.

As used herein, the terms “intracellularly cleaved” and “intracellularcleavage” refer to a metabolic process or reaction inside a cell on anAffinity Medicant Linker conjugate (e.g., an Antibody Medicant conjugate(AMC) or the like), whereby the covalent attachment, e.g., the linker,between the Medicant moiety (M) and the Affinity unit (e.g., an antibody(Ab)) is broken, resulting in the free Medicant, or other metabolite ofthe conjugate dissociated from the antibody inside the cell. The cleavedmoieties of the Affinity Medicant Linker conjugate are thusintracellular metabolites.

As used herein, the term “bioavailability” refers to the systemicavailability (i.e., blood/plasma levels) of a given amount of a medicantadministered to a patient. Bioavailability is an absolute term thatindicates measurement of both the time (rate) and total amount (extent)of medicant that reaches the general circulation from an administereddosage form.

As used herein, the term “cytotoxic activity” refers to a cell-killing,a cytostatic or an anti-proliferative effect of an Affinity MedicantLinker conjugate or an intracellular metabolite of an Affinity MedicantLinker conjugate. Cytotoxic activity may be expressed as the IC₅₀ value,which is the concentration (molar or mass) per unit volume at which halfthe cells survive.

As used herein, the term “cytotoxic agent” as used herein refers to asubstance that inhibits or inhibits the function of cells and/or causesdestruction of cells. The term is intended to include radioactiveisotopes (e.g., ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P,⁶⁰C, and radioactive isotopes of Lu), chemotherapeutic agents, andtoxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including synthetic analogsand derivatives thereof. In one aspect, the term does not include aradioactive isotope(s).

As used herein, a “disorder” is any condition that would benefit fromtreatment with an Affinity Medicant Linker Conjugate. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose a mammal to the disorder in question.Non-limiting examples of disorders to be treated herein include benignand malignant cancers; leukemia and lymphoid malignancies, neuronal,glial, astrocytal, hypothalamic and other glandular, macrophagal,epithelial, stromal and blastocoelic disorders; and inflammatory,angiogenic and immunologic disorders.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition or disorder in mammals that is typicallycharacterized by unregulated cell growth. A “tumor” comprises one ormore cancerous cells.

As used herein, an “autoimmune disease” herein is a disease or disorderarising from and directed against an individual's own tissues or aco-segregate or manifestation thereof or resulting condition therefrom.

As used herein, an example of a “patient” includes, but is not limitedto, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog,cat, bird and fowl. In an exemplary embodiment, the patient is a human.

As used herein, the terms “treat” or “treatment,” unless otherwiseindicated by context, refer to therapeutic treatment and prophylacticmeasures to prevent relapse, wherein the object is to inhibit or slowdown (lessen) an undesired physiological change or disorder, such as thedevelopment or spread of cancer. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already having the condition or disorder as wellas those prone to have the condition or disorder.

As used herein, in the context of cancer, the term “treating” includesany or all of inhibiting growth of tumor cells, cancer cells, or of atumor; inhibiting replication of tumor cells or cancer cells, lesseningof overall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

As used herein, in the context of an autoimmune disease, the term“treating” includes any or all of inhibiting replication of cellsassociated with an autoimmune disease state including, but not limitedto, cells that produce an autoimmune antibody, lessening theautoimmune-antibody burden and ameliorating one or more symptoms of anautoimmune disease.

As used herein, in the context of an infectious disease, the term“treating” includes any or all of inhibiting the growth, multiplicationor replication of the pathogen that causes the infectious disease andameliorating one or more symptoms of an infectious disease.

As used herein, the term “package insert” is used to refer toinstructions customarily included in commercial packages of therapeuticproducts, that contain information about the indication(s), usage,dosage, administration, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, a “native sequence” polypeptide is one which has thesame amino acid sequence as a polypeptide, e.g., a tumor-associatedantigen receptor, derived from nature. Such native sequence polypeptidescan be isolated from nature or can be produced by recombinant orsynthetic means. Thus, a native sequence polypeptide can have the aminoacid sequence of a naturally-occurring human polypeptide, a murinepolypeptide, or a polypeptide from any other mammalian species.

As used herein, an “isolated” nucleic acid molecule is a nucleic acidmolecule that is identified and separated from at least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the nucleic acid. An isolated nucleic acid molecule isother than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from thenucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that ordinarily express the nucleic acid where, forexample, the nucleic acid molecule is in a chromosomal locationdifferent from that of natural cells.

As used herein, the expression “control sequences” refers to nucleicacid sequences necessary for the expression of an operably linked codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

As used herein, a nucleic acid is “operably linked” when it is placedinto a functional relationship with another nucleic acid sequence. Forexample, DNA for a pre-sequence or secretory leader is operably linkedto DNA encoding a polypeptide if it is expressed as a pre-protein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence, for example, if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the DNA sequencesbeing linked are contiguous, and, in the case of a secretory leader,contiguous and in reading phase. However, enhancers do not have to becontiguous. Linking can be accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers can be used in accordance withconventional practice.

As used herein, the terms “cell,” “cell line,” and “cell culture” areused interchangeably and all such designations include progeny. Thewords “transformants” and “transformed cells” include the primarysubject cell and cultures or progeny derived therefrom without regardfor the number of transfers. It is also understood that all progeny maynot be precisely identical in DNA content, due to deliberate orinadvertent mutations. Mutant progeny that have the same function orbiological activity as screened for in the originally transformed cellare included. Where distinct designations are intended, it will be clearfrom the context.

As used herein, the term “chiral” refers to molecules which have theproperty of non-superimposability of the mirror image partner, while theterm “achiral” refers to molecules which are superimposable on theirmirror image partner.

As used herein, the term “stereoisomers” refers to compounds which haveidentical chemical constitution, but differ with regard to thearrangement of the atoms or groups in space.

As used herein, “diastereomer” refers to a stereoisomer with two or morecenters of chirality and whose molecules are not mirror images of oneanother. Diastereomers have different physical properties, e.g., meltingpoints, boiling points, spectral properties, and reactivities. Mixturesof diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e., C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs andisomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and thelike. An unsaturated alkyl group is one having one or more double bondsor triple bonds. Examples of unsaturated alkyl groups include, but arenot limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy isan alkyl attached to the remainder of the molecule via an oxygen linker(—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred in the presentinvention. A “lower alkyl” or “lower alkylene” is a shorter chain alkylor alkylene group, generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, consisting of at least one carbon atom and atleast one heteroatom selected from the group consisting of O, N, P, Si,and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂—and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR, —SR′, and/or —SO₂R′. Where “heteroalkyl”is recited, followed by recitations of specific heteroalkyl groups, suchas —NR′R″ or the like, it will be understood that the terms heteroalkyland —NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted herein as excluding specificheteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,and 3-bromopropyl.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain from one to four heteroatoms selected from N, O, and S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl”includes fused ring heteroaryl groups (i.e., multiple rings fusedtogether wherein at least one of the fused rings is a heteroaromaticring). A 5,6-fused ring heteroarylene refers to two rings fusedtogether, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl, and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxyl)propyl, and the like).

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfoxide” as used herein, means a moiety having theformula R—S(O)—R′, where R and R′ are alkyl groups as defined above. Rand R′ may have a specified number of carbons (e.g., “C₁-C₄alkylsulfoxide”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR′″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″, and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound ofthe invention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″, and R″″ groupwhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″′, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″, and R″″ are preferably independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ groups when more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties: (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, substituted with at least onesubstituent selected from: oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂,halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, andunsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that are prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included are salts of amino acids such as arginate, and saltsof organic acids like glucuronic or galacturonic acids. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds in aprodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds of the present invention.Additionally, prodrugs can be converted to the compounds of the presentinvention by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to the compounds of thepresent invention when placed in a transdermal patch reservoir with asuitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers, and individual isomers are encompassedwithin the scope of the present invention. The compounds of the presentinvention do not include those that are known in the art to be toounstable to synthesize and/or isolate.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), carbon-13 (¹³C), or carbon-14 (¹⁴C). Allisotopic variations of the compounds of the present invention, whetherradioactive or not, are encompassed within the scope of the presentinvention.

Many organic compounds exist in optically active forms, i.e., they havethe ability to rotate the plane of plane-polarized light. In describingan optically active compound, the prefixes D and L, or R and S, are usedto denote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or 1 meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesestereoisomers are identical except that they are mirror images of oneanother. A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

As used herein, an amino acid “derivative” includes an amino acid havingsubstitutions or modifications by covalent attachment of a parent aminoacid, such as, e.g., by alkylation, glycosylation, acetylation,phosphorylation, and the like. Further included within the definition of“derivative” is, for example, one or more analogs₁ of an amino acid withsubstituted linkages, as well as other modifications known in the art.

As used herein, a “natural amino acid” refers to arginine, glutamine,phenylalanine, tyrosine, tryptophan, lysine, glycine, alanine,histidine, serine, proline, glutamic acid, aspartic acid, threonine,cysteine, methionine, leucine, asparagine, isoleucine, and valine,unless otherwise indicated by context.

As used herein, a “protecting group” refers to a moiety that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Representative hydroxy protecting groups include acylgroups, benzyl and trityl ethers, tetrahydropyranyl ethers,trialkylsilyl ethers and allyl ethers. Representative amino protectinggroups include formyl, acetyl, trifluoroacetyl, benzyl,benzyloxycarbonyl (CBZ), tert-butoxycarbonyl (Boc), trimethyl silyl(TMS), 2-trimethylsilyl-ethanesulfonyl (SES), trityl and substitutedtrityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC),nitro-veratryloxycarbonyl (NVOC), and the like. Examples of a “hydroxylprotecting group” include, but are not limited to, methoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ether, benzyl ether,p-methoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,triisopropyl silyl ether, t-butyldimethyl silyl ether, triphenylmethylsilyl ether, acetate ester, substituted acetate esters, pivaloate,benzoate, methanesulfonate and p-toluenesulfonate.

Abbreviations used include: DMAP=4-dimethylaminopyridine;DCC=N,N′-dicycyclohexylcarbodiimide;ODHBt=3,4,-dihydroxy-4-oxo-1,2,3-benzo-triazine-3-yl ester;NMM=N-methylmorpholin; EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; DIAD=diisopropyl azodicarboxylate; DEAD=diethylazodicarboxylate; and DIPC=N,N′-diisopropylcarbodiimide.

As used herein, a “leaving group” refers to a functional group that canbe substituted by another functional group. Such leaving groups are wellknown in the art, and examples include, but are not limited to, a halide(e.g., chloride, bromide, and iodide), methanesulfonyl (mesyl),p-toluenesulfonyl (tosyl), trifluoromethylsulfonyl (triflate), andtrifluoromethylsulfonate.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound(e.g., a Medicant Linker compound, or an Affinity Medicant Linkerconjugate). The compound typically contains at least one amino group,and accordingly acid addition salts can be formed with this amino group.Exemplary salts include, but are not limited to, sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and palmoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counter ion.The counter ion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counter ion.

As used herein, a “pharmaceutically acceptable solvate” or “solvate”refer to an association of one or more solvent molecules and a compoundof the invention, e.g., an Affinity Medicant Linker conjugate or aMedicant Linker compound. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

The following abbreviations are used herein and have the indicateddefinitions: Boc is N-(t-butoxycarbonyl), cit is citrulline, dap isdolaproine, DCM is dichloromethane, DIEA is N,N-diisopropylethylamine,dil is dolaisoleuine, DMF is N,N-dimethylformamide, DMSO isdimethylsulfoxide, doe is dolaphenine, dov is N,N-dimethylvaline, DTNBis 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HBTU is2-[1H-benzotriazole-1-yl]-1,1,3,3-tetramethylaminiumhexafluorophosphate; HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeOHis methanol, MeVal is N-methyl-valine, PAB is p-aminobenzyl, PBS isphosphate-buffered saline (pH 7.4), Ph is phenyl, phe isL-phenylalanine, PyBrop is bromo tris-pyrrolidino phosphoniumhexafluorophosphate, TFA is trifluoroacetic acid, UV is ultraviolet, andval is valine.

The following LU abbreviations are used herein and have the indicateddefinitions: Val Cit or vc is a valine-citrulline dipeptide site inprotease cleavable linker; PABC is p-aminobenzylcarbamoyl; (Me)vc isN-methyl-valine citrulline, where the linker peptide bond has beenmodified to prevent its cleavage by cathepsin B; and MC(PEG)₆-OH ismaleimidocaproyl-polyethylene glycol.

As used herein, a “pegylated compound” refers to a compound conjugatedwith two or more polyethylene glycol moieties or two or morepolypropylene glycol moieties or a combination thereof.

As used herein, a “pro-peptide” includes pro-peptide, pre-peptide,pro-protein and pre-protein amino acid sequences including those aminoacid sequences cleaved by enzymes disclosed in Table IX.

As used herein, “Illudin1” or “illudin-1” means those analogs disclosedin Table XI. As used herein “Illudin2” or “illudin2” means those analogsdisclosed in Table XI and Table XII. As used herein, “acylfulvene” means“illudin2” and any analog derived therefrom.

Malignant neoplasia is the second most common cause of death in theUnited States behind cardiovascular disease. Chemotherapy has exerted apredominant role in increasing life spans for patients with a variety oftumors including Burkitt's lymphoma, acute lymphocytic leukemia andHodgkin's disease. Further, new cancer chemotherapeutic agents andmethods of care combined with early detection and treatment haveresulted in decreases in the overall incidence of cancer and decreasesin the death rates from all cancers combined. Responsive tumorsrepresent only a small fraction of the various types of cancer. Further,agents such as cyclophosphamide, adriamycin, 5-fluorouracil andhexamethylmelamine, which are highly active against clinical solidtumors, are limited. Thus, patients with many types of malignanciesremain at significant risk for relapse and mortality. After relapse,some patients can be re-induced into remission with their initialtreatment regimen. However, higher doses of the initial chemotherapeuticagent or the use of additional agents are frequently required,indicating the development of at least partial medicant resistance.Evidence indicates medicant resistance can develop simultaneously toseveral agents, including medicant resistance to treatments to which thepatient was not exposed. The development of multiple-medicant resistanttumors may be a function of tumor mass and constitutes a major cause oftreatment failure. To overcome this medicant resistance, high-dosechemotherapy with or without radiation and allogenic or autologous bonemarrow transplantation can be employed. The high-dose chemotherapy mayemploy the original medicant(s) or be altered to include additionalagents. As a result, there remain many cancer patients for whom no orminimally effective therapy exists. Accordingly, there is a need for thedevelopment of novel chemotherapeutics with greater efficacy or safety,either as monotherapy or in combination with other chemotherapeuticagents, and such agents with the potential to overcome medicantresistance in cancer cells.

Illudins are toxic natural products produced by mushrooms of the genusOmphalotus (FIG. 22A). Syn-Illudins are semi-synthetic derivatives ofIlludins. Acylfulvenes are also semi-synthetic derivatives of Illudins.Syn-Illudins and Acylfulvenes have each been chemically modified atselect sites to allow their use as medicants. The modifications in theSyn-Illudins do not alter any of the cyclic rings (cyclopropane,cyclopentane, cyclohexane) of the basic Illudin chemical structure (FIG.22B). The modifications of Acylfulvenes differ from Syn-Illudins in thatan additional double bond (an unsaturated bond) has been created in the5 membered (cyclopentane) ring (FIG. 22C, FIG. 22D).

Illudins function as alkylating agents that damage DNA and thereby blocktranscription. The blockage can be repaired through nucleotide excision.The toxicity of the illudins has prevented any applications in humantumor therapy. Acylfulvenes have been developed which exhibit promisingantitumor activity with a better safety profile, as described in U.S.Pat. Nos. 5,439,936; 5,523,490 and 6,380,403 which are each hereinexpressly incorporated by reference in their entireties. Irofulven or6-hydroxymethylacylfulvene (see FIG. 6) is an analog of illudin S whichhas demonstrated clinical activity with an acceptable safety profile inhormone-refractory prostate cancer. Most relevant to clinicalapplications, irofulven activity is independent of common resistancemechanisms such as the multi-medicant resistance phenotype,anti-apoptotic B-cell lymphoma 2 (Bcl-2) (SEQ. ID. 056) over expression,as well as tumor protein 53 (p53) (SEQ. ID. 057) and cyclin dependentkinase inhibitor 1 (p21/WAF1) (SEQ. ID. 058) mutations (see FIG. 7 andTable XIV).

Growth factors, including peptides and proteins are critical mediatorsof a wide range of cell-cell communication. They are importantendocrine, paracrine and autocrine messengers. Growth factors functionas neurotransmitters and neuromodulators, regulate chemotaxis, immunefunction, development, cell growth, and can influence tumor cells. Thereceptors that recognize growth factors are highly selective and definespecific cell populations. As a result, growth factor receptors are alarge and important class of medicant (including drug) targets. Inaddition to physiologic noncancerous cell populations, these receptorscan also be expressed in various cancer cell populations.

A polypeptide is a long, continuous, and unbranched chain of aminoacids. A glycol-peptide is a peptide that contains one or morecarbohydrate moieties covalently attached to the side chains of specificamino acids. A pro-peptide, is an inactive peptide that can be turnedinto an active form through a post translational modification thatenzymatically cleaves the pro-peptide. Examples include pro-insulin(SEQ. ID. 059) and pro-opiomelanocortin (SEQ. ID. 060). Enzymaticallycleaving the pro-peptide, allows for the peptide to be available onshort notice and/or in large quantities. Some pro-peptides are secretedfrom the cell. Many of these are synthesized with an N-terminal signalpeptide that targets the pro-peptide for secretion.

Cytokines are small proteins (approximately 5 to 20 kDa) that affect thebehavior of other cells, and sometimes the releasing cell itself and arethereby important in cell signaling (see Table XIV). Many specificcytokines can be released by a variety of different kinds of cells,e.g., macrophages, B lymphocytes, T lymphocytes, mast cells, endothelialcells, fibroblasts, and various stromal cells. Cytokines act throughspecific receptors, and are important in the humoral and cell-basedimmune responses. Cytokines also regulate the maturation, growth, andresponsiveness of specific cell populations. Cytokines circulate in muchhigher concentrations than hormones and in contrast with hormones aremade by a variety of different kinds of cells. Cytokines are importantin host responses to infection, immune responses, inflammation, trauma,sepsis, cancer, and reproduction. As a result, cytokine receptors areupregulated in many forms of cancers.

A steroid is an organic compound that contains four cycloalkane ringsjoined to each other. Examples of steroids include the dietary lipidcholesterol and the sex hormones estradiol and testosterone. The core ofa steroid molecule is composed of seventeen carbon atoms bonded togetherthat take the form of four fused rings: three six-carbon atom rings andone five-carbon atom ring. A variety of functional groups can beattached to the four-ring core. Steroids can also vary depending on theoxidation state of the rings. A steroid hormone is a steroid that actsas a hormone. Steroid hormones can be grouped into five groups(glucocorticoids, mineralocorticoids, androgens, estrogens, andprogesterones) based on the receptors to which they bind. Steroidhormones, particularly androgens, are essential not only for growth anddevelopment but also in the progression of many forms of cancer. As aresult, steroid hormone receptors are upregulated in many forms ofcancers.

The retinoic acid receptor (RAR) is a nuclear receptor which can alsoact as a transcription factor. The RAR can be activated by eitherall-trans retinoic acid or 9-cis retinoic acid. There are three RARisoforms (alpha (SEQ. ID. 061), beta (SEQ. ID. 062), and gamma (SEQ. ID.063)), each encoded by separate genes, where splice variants generatestill further diversity in the expressed receptor. The retinoid Xreceptor (RXR) is a nuclear receptor activated by 9-cis retinoic acid.There are also three RXR isoforms (alpha (SEQ. ID. 064), beta (SEQ. ID.065), and gamma (SEQ. ID. 066)), each encoded by separate genes. RXRhetero-dimerizes with subfamily 1 nuclear receptors including RAR. Inthe absence of ligand, the RAR/RXR dimer binds to retinoic acid responseelements complexes with a co-repressor protein. Binding of agonistligands to RAR results in dissociation of the co-repressor andrecruitment of a co-activator protein that, in turn, promotestranscription of the downstream target gene into mRNA and therebyprotein or other RNA signaling mechanisms.

Lipid metabolism is altered in many forms of cancer, includingupregulation of de novo lipid synthesis. Cancer cells can also usealternative enzymes and pathways to facilitate the production of fattyacids. These newly synthesized lipids may support a number of cellularprocesses to promote cancer cell proliferation and survival. Elaidicacid or (E)-octadec-9-enoic acid is the trans isomer of oleic acid andis found in small quantities in caprine milk, bovine milk and somemeats. It increases Cholesteryl Ester Transfer Protein (CETP) (SEQ. ID.067) activity, which in turn raises levels of very low densitylipoprotein and lowers levels of high density lipoprotein (HDL)cholesterol. CETP is found in plasma, where it is involved in thetransfer of cholesteryl ester from HDL to other lipoproteins. Defects inthe CETP gene are a cause of hyperalphalipoproteinemia 1.

An antibody is a protein made up of four peptide chains disulfide linkedtogether to form a “Y”-shape. Antibodies are produced by plasma cellsand are used by the immune system to identify and neutralize foreignantigens such as bacteria and viruses. The antibody recognizes a uniquepart of the antigen using each FAB portion of the protein (i.e., the tipof the “Y” portion of the antibody), allowing a specific high affinitybinding interaction to occur. The binding interaction of differentantibodies can target specific antigen epitopes. An antibody fragmentcontaining one or both FAB portions can also target specific antigenepitopes.

The ability of the Illudin, Syn-Illudin and Acylfulvene analogs toinhibit tumor cell growth is shown in Table XV. The MV522 cell line is alung-derived adenocarcinoma cell line. In various embodiments of theinvention, the MV522 cell line represents a “target” cell line. That isan Illudin, Syn-Illudin or Acylfulvene analog that exhibits toxicityagainst this solid tumor cell line shows a desirable result. The 8392Bcell line represents a hematopoietic (non-solid) cell line. In variousembodiments of the invention, the 8392B cell line is considered a“nontarget” cell line. The two hour toxicity data represents theconcentration of a given analog for which a two hour exposure willinhibit 50% of the DNA synthesis activity in a given cell line. The 48hour exposure data represents the concentration at which a given analogwith a 48 hour exposure will inhibit the growth or viability in a givencell line as defined by the standard Trypan Blue Exclusion assay. As anexample, analog 002 (FIG. 20AB) will inhibit the target MV522 cell lineat 110 nM with only a 2 hour exposure but has no inhibitory effect onthe nontarget 8392B cell line at 26,000 nM (26 μM). Analog 002 with aprolonged exposure period (e.g. 48 hours) can eventually inhibit thenontarget cell line. In contrast, Analog 201 (FIG. 20HR) will inhibitthe target. MV522 cell line with only a 2 hour exposure (IC50=360 nM)but has minimal effect on the 8392B cell nontarget line with even a 48hour exposure (IC50=26,0000 nM) indicating superior anticancer activityas a monotherapeutic agent. In contrast to these two analogs, analog 224(FIG. 20IO) displayed minimal toxicity as well as no differentialtoxicity between the target and nontarget cell line indicating it wouldhave minimal properties as a monotherapeutic anticancer agent.

As used herein, a “growth factor” or an “anti-angiogenic protein”includes Adrenomedullin (SEQ. ID. 068), Angiopoietin (Ang) (SEQ. ID.069, 106, 111, and 145), Autocrine motility factor (SEQ. ID. 070), Bonemorphogenetic proteins (BMPs) (SEQ. ID. 071), Brain-derived neurotrophicfactor (BDNF) (SEQ. ID. 072), Endostatin (SEQ. ID. 073), Endostar (SEQ.ID. 074), Epidermal growth factor (EGF) (SEQ. ID. 075), Erythropoietin(EPO) (SEQ. ID. 076), Fibroblast growth factor (FGF) (SEQ. ID. 077),Glial cell line-derived neurotrophic factor (GDNF) (SEQ. ID. 078),Granulocyte colony-stimulating factor (G-CSF) (SEQ. ID. 079),Granulocyte macrophage colony-stimulating factor (GM-CSF) (SEQ. ID.080), Growth differentiation factor-9 (GDF9) (SEQ. ID. 081), Hepatocytegrowth factor (HGF) (SEQ. ID. 082), Hepatoma-derived growth factor(HDGF) (SEQ. ID. 083), Insulin-like growth factor (IGF) (SEQ. ID. 084),Migration-stimulating factor (SEQ. ID. 085), Myostatin (GDF-8) (SEQ. ID.086), Nerve growth factor (NGF) (SEQ. ID. 087) and other neurotrophins(SEQ. ID. 144), Platelet-derived growth factor (PDGF A) (SEQ. ID. 088),PDGF B (SEQ. ID 168), PDGF C (SEQ. ID 036), PDGF D (SEQ. ID 037),Thrombopoietin (TPO) (SEQ. ID. 089), Transforming growth factoralpha(TGF-α) (SEQ. ID. 090), Transforming growth factor beta(TGF-β)(SEQ. ID. 091), Tumor necrosis factor-alpha(TNF-α) (SEQ. ID. 092),Vascular endothelial growth factor (VEGF) (SEQ. ID. 093), and placentalgrowth factor (PlGF) (SEQ. ID. 094).

As used herein, a “protein toxin” includes ricin A chain (SEQ. ID. 095),ricin B chain (SEQ. ID. 096), diphtheria toxin (SEQ. ID. 097),Pseudomonas aeurginosa exotoxin A (SEQ. ID. 098), r-gelonin (SEQ. ID.099), saporin (SEQ. ID. 100), glycosylated protein toxins, deglcosylatedprotein toxins and protein toxin fragments which includes deglycosylatedricin A, deglycosylated ricin B, Pseudomonas aeurginosa exotoxin A PE40fragment (SEQ. ID. 101) and Pseudomonas aeurginosa exotoxin A PE38fragment (SEQ. ID. 102).

As used herein, a “steroid” includes cholesterol (5-cholesten-3beta-ol),pregnenolone (3beta-hydroxy-5-pregnen-20-one), 17-hydroxyprenenolone(3-beta,17-dihydroxy-5-pregnen-20-one), progesterone(4-pregnene-3,20-dione), 17-hydroxyprogesterone(17-hydroxy-4-pregnene-3,20-dione), androstenedione(4-androstene-3,17-dione), 4-hydroxyandrostenedione(4-hydroxy-4-androstene-3,17-dione), 11-beta-hydroxyandostenedione(11beta-4-androstene-3,17-dione), androstanediol(3-beta,17-beta-Androstanediol), androsterone(3-alpha-hydroxy-5alpha-androstan-17-one), epiandrosterone(3-beta-hydroxy-5alpha-androstan-17-one), adrenosterone(4-androstene-3,11,17-trione), dehydroepiandrosterone(3beta-hydroxy-5-androsten-17-one), dehydroepiandrosterone sulfate(3-beta-sulfooxy-5-androsten-17-one), testosterone(17beta-hydroxy-4-androsten-3-one), epitestosterone(17-alpha-hydroxy-4-androsten-3-one), 5-alpha-dihydrotesterone(17-beta-hydroxy-5alpha-androstan-3-one), 5-beta-dihydrotestosterone(17-beta-hydroxy-5beta-androstan-3-one), 11-beta-hydroxytestosterone(11-beta,17beta-dihydroxy-4-androsten-3-one), 11-ketotesosterone(17-beta-hydroxy-4-androsten-3,17-dione), estrogen (including: estrone(3-hydroxy-1,3,5(10)-estratrien-17-one), estradiol(1,3,5(10)-estratriene-3,17beta-diol), and estriol(1,3,5(10)-estratriene-3,16alpha,17beta-triol)), corticosterone(11-beta,21-dihydroxy-4-pregnene-3,20-dione), deoxycorticosterone(21-hydroxy-4-pregnene-3,20-dione), cortisol(11-beta,17,21-trihydroxy-4-pregnene-3,20-dione), 11-deoxycortisol(17,21-dihydroxy-4-pregnene-3,20-dione), cortisone (17,21-dihydroxy-4-pregnene-3, 11,20-trione), 18-hydroxycorticosterone(11-beta,18,21-trihydroxy-4-pregnene-3,20-dione),1-alpha-hydroxycorticosterone(1-alpha,11-beta,21-trihydroxy-4-pregnene-3,20-dione), and aldosterone(18,11-hemiacetal of 11beta,21-dihydroxy-3,20-dioxo-4-pregnen-18-al).

As used herein, a “Specific Binding Peptide” includes an“anti-angiogenic peptide” (SEQ. ID. 146) and an “integrin bindingpeptide” (SEQ. ID. 147). A “Specific Binding Peptide” includes integrinbinding peptide RGD4C=CDCRGDFC (SEQ. ID. 147), integrin binding peptideRGD10 (SEQ. ID. 148), c(RGDyK) (SEQ. ID. 149), integrin binding peptidec(RGDfK) (SEQ. ID. 150), integrin binding peptide [c(RGDyK)]2 (SEQ. ID.151), integrin binding peptide CAGKNFFWKTFTSC (SEQ. ID. 152),cilengitide (cyclic RGD pentapeptide) (SEQ. ID. 153), ATN-161 (peptideantagonist of integrin alpha5betal) (SEQ. ID. 154), ATN-454(Ac-PHSCN—NH₂) (peptide antagonist of integrin alpha5betal) (SEQ. ID.155), tumstatin T7 peptide TMPFLFCNVNDVCNFASRNDYSYWL (SEQ. ID. 156),tumstatin sequence 1 YSNS (SEQ. ID. 157), tumstatin sequence 2 YSNSG(SEQ. ID. 158), endostatin motif FLSSRLQDLYSIVRRADRAA (SEQ. ID. 159),endostatin motif IVRRADRAAVP (SEQ. ID. 160), laminin peptide A13(RQVFQVAYIIIKA) (SEQ. ID. 161), laminin peptide C16 (KAFDITYVRLKF) (SEQ.ID. 162), laminin peptide C16S (DFKLFAVTIKYR) (SEQ. ID. 163), and VEGFR1peptide (CPQPRPLC) (SEQ. ID. 164).

As used herein, a traditional linker includes linkers that can be formedfrom those reagents disclosed in Tables IA-ID, IIA-IID, IIIA-IIIC,IVA-IVC, VA-VB, and VIA-VID.

As used herein, a “FSB linker” includes those linkers selected from thegroup consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyland 2-fluorosulfonyl benzoyl as depicted in FIG. 15.

As used herein, a “Mal1” linker includes a malonic linker and amaleimide linker covalently attached to an Illudin, Syn-Illudin, orAcylfulvene (FIG. 20HF, FIG. 20HG, FIG. 20HO).

As used herein, a “protease” includes those enzymes disclosed in TableIX.

As used herein, a “cytokine” includes chemokines, interferons,interleukins, lymphokines, tumor necrosis factor, neutrophil activatingprotein-2, and monocyte chemotactic protein-1 and those compoundsdisclosed in Table XIV.

Despite recent advances in therapy, many patients with cancer invariablyrelapse and require additional treatments. Most of these patient'scancers become refractory to standard chemotherapy and/or radiationtreatment regimens. The prognosis for these patients is poor and longterm survival rates for metastatic solid tumor cancers remain very low.Thus, there is a need for the development of novel agents and treatmentregimens that specifically target these recurring tumor cells and alsoproduce less systemic toxicity. Target therapies, such as monoclonalantibodies, now provide a promising alternative to the conventionalcytotoxic chemotherapy approach.

Monoclonal antibody based therapy has recently achieved considerablesuccess in oncology and there are currently nine monoclonal antibodies(without a medicant attached) approved by the FDA as cancertherapeutics. As an example, HERCEPTIN® and RITUXAN® (both produced byGenentech, South San Francisco, Calif.), are used to successfully treatbreast cancer and non-Hodgkin's lymphoma, respectively. HERCEPTIN® is arecombinant DNA-derived humanized monoclonal antibody selectivelybinding to the extracellular domain of the Human Epidermal growth factorReceptor 2 (HER2) proto-oncogene whereas RITUXAN® is a geneticallyengineered chimeric murine/human monoclonal antibody directed againstthe CD20 antigen overexpressed on the surface of normal and malignant Blymphocytes.

Recent clinical evidence indicates that while the monoclonal antibodybased therapies are effective at inducing remission, they do not alwaysproduce a complete cure, and relapses eventually occur in most patients.There is now a tremendous interest in the use of antibody medicantconjugates as a class of therapeutics that utilize theantigen-selectivity of monoclonal antibodies to deliver potent cytotoxicmedicants to specific tumor cells. Antibody medicant conjugates areproduced by attaching a cytotoxic agent to an antibody that bindsspecifically to a tumor-associated antigen.

In theory, antibody medicant conjugates can confer an increasedtherapeutic index to highly potent medicants by improving therapeuticefficacy and reducing systemic toxicity (by minimizing damage to normaltissues), although this goal has been elusive in achieving. The basisfor the efficacy of antibody medicant conjugates is that they targettumor cells that preferentially express an antigen that is recognized bythe associated antibody. In contrast, non-tumor cells either fail toexpress this antigen, or express the antigen at a very low level. Intheory, only the tumor cells expressing the associated antibody arerecognized and destroyed by the AMC, and other cells are left untouchedand undamaged.

While different medicant classes have been tried for delivery viaantibodies, only a few have proved efficacious for use as antibodymedicant conjugates. The two main medicant classes used to date toproduce antibody medicant conjugates are the auristatins(MMAE/N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine orMMAF/N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) andthe maytansines (DM1 or DM4). Currently only two antibody medicantconjugates are approved by the U.S.F.D.A. and marketed; brentuximabvedotin (auristatin based) and ado-trastuzumab emtansine (maytansinebased).

Illudins (see FIG. 22A, where R═CH₃OH or OH), Syn-illudins (see FIG.22B, where X or Y═C, N, S, O and Z═O or NH or NOH), and Acylfulvenes(see FIG. 22C and FIG. 22D, where X═C, N, S, O and n>1) have severalunique properties over agents traditional used to make antibody drugconjugates (ADCs). Firstly, these are the only agents known to functionby inhibition of the DNA transcription-coupled repair pathway (see FIG.5). No other toxin, drug or medicant inhibits this pathway. The resultis that Illudins, Syn-illudins, and Acylfulvenes are true cytotoxicagents whereas other agents traditionally used to produce ADCs(pyrrolobenzodiazepines, maytansines, fumagillols, dolstatins,auristatins, enadiynes, halichondrins, and tubulysins) are onlycytostatic. In the NCI-DTP 60 cell line panel these other agents werecapable of inhibiting tumor cell growth (IC₅₀ value), had some abilityto block tumor cell growth (TGI value) but none were capable of actuallycausing tumor cell death or cytotoxicity (Table XIII). The illudinderivatives, however, are capable of killing tumor cells at nanomolarconcentrations (Table XIII). This means that while ADCs developed usingother toxins can stall tumor cell growth, they cannot actually kill thetumor cell. Once the effect of the drug has worn off the tumor cellswill again grow and kill the patient. In contrast, the Illudins,Syn-illudins, and Acylfulvenes actually kill the tumor cell with aslittle as a 2 hour exposure (see FIG. 4). Secondly, whereas tumor cellswill undergo apoptosis or cell death with hours once the DNAtranscription-coupled repair pathway is blocked, normal diploidnon-tumor cells can survive for hours. This translates into a widetherapeutic window for ADCs developed with Illudins, Syn-illudins, andAcylfulvenes. The two ADC agents currently FDA approved foradministration deliver a dose of the associated toxin that is 300%higher than a lethal dose which is why these agents have severe systemictoxicity. In contrast, the comparable ADC developed with Illudins,Syn-illudins, or Acylfulvenes will deliver a dose of the associatedtoxin that is 40% of a known non-toxic dose (estimated at 28% of a toxicdose and only 12% of a lethal dose). Thus, ADCS developed with Illudins,Syn-illudins, and Acylfulvenes will have minimal systemic toxicity ascompared to current agents. Thirdly, these agents are stable down to apH of 2.0. An ADC is engulfed by a tumor cell, transported to theendosomes (pH<6.0) and then into the lysozomes (pH<4). Many agents usedfor ADCs will degrade in these low pH environment, whereas Illudins,Syn-illudins, and Acylfulvenes are stable. 4) Cancer cells can becomeresistant to various toxins and drugs through the development of what istermed multi-drug resistance. This process is known to occur throughseveral different mechanisms. Whereas other toxins and drugs aresubstrates for the most common MDR mechanisms (MDR1/gp170 andMRP/gp180), and cancer cells can become resistant to these agents, theIlludins, Syn-illudins, and Acylfulvenes remain active against all MDRphenotypes regardless of the mechanism (see FIG. 7 and Table XIV).Hence, if tumor cells have already developed multi-drug resistance priorto ADC with a conventional toxin, or during the administration of acourse of the ADC, the ADC will have no efficacy. In contrast, ADCsdeveloped with Illudins, Syn-illudins, or Acylfulvenes will continue tokill cancer cells.

The present invention is based on the unexpected discovery thatacylfulvenes, can be conjugated directly to a linker, via a variety ofpeptide or non-peptide bonds, and are active as medicant delivery agentsin vitro and in vivo. Similar to other medicant classes used to produceantibody medicant conjugates, the acylfulvenes can be conjugated to alinker that allows subsequent coupling to a monoclonal antibody.Surprisingly, unlike previous medicant classes such as the auristatins(MMAE, MMAF, dolstatin-10), the maytansines (DM1 or DM4), theirinotecans and their metabolites (SN38), the calicheamicins (17-DMAG),the pyrrolobenzodiazepines (SJG-136), the duocarmycins (CC-1065), manyof the acylfulvene compounds do not require a linker and can be directlyattached to a monoclonal antibody or fragment thereof by a variety ofsimple chemical reactions. In this sense, the lack of requirement for alinker or a spacer, the acylfulvene compounds are unique. They willdirectly form covalent bonds with reactive groups on an AM such as amonoclonal antibody. In addition, because of their very small size andextreme cytotoxicity the acylfulvenes can be coupled directly to verysmall molecular weight entities (or affinity moieties) that allow tumorspecific cytotoxicity without the concomitant requirement of use of amonoclonal antibody. Examples include the ability to linkilludins/acylfulvenes directly to steroids which allow themedicant-affinity complex to kill cells overexpressing a specificsteroid receptor (such as estrogen- or progesterone-positive breastcancer cells) or even to be chemically coupled to various lipids. Thesmall size and extreme cytotoxicity acylfulvenes allows direct couplingto peptides which can preferentially bind to tumor cells (integrinbinding peptides) or display anti-angiogenic properties to hinder tumorinvasion. The illudins/acylfulvenes can also be coupled to specificpeptides which actually renders the medicant-affinity complex non-toxicuntil the peptide is cleaved by a protease secreted by tumor cells. Anexample includes PSA (prostate specific antigen) secreted by prostateadenocarcinoma cells. Again, unlike previous medicant classes such asthe auristatins (MMAE, MMAF, dolstatin-10), the maytansines (DM1 orDM4), the irinotecans and their metabolites (SN38), the calicheamicins(17-DMAG), the pyrrolobenzodiazepines (SJG-136), the duocarmycins(CC-1065), the acylfulvene compounds do not require a linker and can bedirectly attached to a steroid or a peptide that will subsequentlyfunction as an AM and direct the associated complex to specific tumorcells. An acylfulvene is attached to either a Specific Binding Peptideor a peptide which if cleaved by a specific protease (see Table IX) suchas PSA generates an entity which is cytotoxic (see Table VIII).

Trastuzumab emtansine (Genentech for Breast cancer) uses maytanasinederive DM-1, a stable non-cleavable linker. Brentuximab vedotin (SeattleGenetic s/Takeda for Hodgkin's Lymphoma) uses auristatin MMAE toanti-CD30, an enzyme sensitive cleavable linker.

The malonic linker, maleimide linker and SMCC [succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate] linker can form activeintermediates that react with sulfhydryl groups on an antibody. SMCC hasbeen used to bind maytansine derivative DM1 to the monoclonal antibodyHerceptin. The AMC was internalized where the Herceptin was degraded byproteases and DM1 was released into the cytosol. Further, Sulfo-SMCC[sulfosuccinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene] formsan active intermediate that reacts with sulfhydryl groups on anantibody. The resulting Sulfo-SMCC AMC is more water soluble than theSMCC AMC.

Compounds and Conjugates. The present invention is drawn to a series ofcompounds and conjugates containing a Medicant moiety (M) linked via itsC terminus to a LU (LU). The LU can operate to provide a suitablerelease of M.

In one group of embodiments, the invention provides Medicant Linkercompounds having Formula I: LU-M (I) or a pharmaceutically acceptablesalt or solvate thereof where the medicant loading is represented by p,the average number of medicant molecules per affinity (e.g., anantibody) (e.g. of Formula II, IIa, IIa′). Medicant loading may rangefrom 1 to 20 Medicant units (M) per Affinity unit (e.g., Ab or in Ab).Compositions of Formula IIa and Formula IIa′ include mixtures ofantibodies conjugated with a range of medicants, from 1 to 20.

In some embodiments, p is from about 1 to about 8 Medicant units perAffinity unit. In some embodiments, p is 1. In some embodiments, p isfrom about 2 to about 8 Medicant units per Affinity unit. In someembodiments, p is from about 2 to about 6, 2 to about 5, or 2 to about 4Medicant units per LU. In some embodiments, p is about 2, about 4, about6 or about 8 Medicant units per Affinity unit.

The average number of Medicants units per Affinity unit in a preparationfrom a conjugation reaction may be characterized by conventional meanssuch as mass spectroscopy, ELISA assay, and HPLC. The quantitativedistribution of Affinity Medicant Linker conjugates in terms of p mayalso be determined. In some instances, separation, purification, andcharacterization of homogeneous Affinity Medicant Linker conjugates,where p is a certain value from Affinity Medicant Linker conjugates withother medicant loadings may be achieved by means such as reverse phaseHPLC or electrophoresis.

Returning to Formula IIa′, the conjugates comprise an antibodycovalently attached to one or more Medicant units (moieties) via a LU:A, a, W and w are as described above. The antibody medicant conjugateinclude pharmaceutically acceptable salts or solvates thereof.

The medicant loading is represented by p, the average number of Medicantunits per antibody in a molecule of Formula II. Medicant loading mayrange from 1 to 20 medicants (M) per Ab or mAb. Compositions of the AMCof Formula IIa′ include mixtures of antibodies conjugated with a rangeof medicants, from 1 to 20. In some embodiments, p is from about 1 toabout 8 Medicant units per antibody. In some embodiments, p is 1. Insome embodiments, p is from about 2 to about 8 Medicant units perantibody. In some embodiments, p is from about 2 to about 6, 2 to about5, or 2 to about 4 Medicant units per antibody. In some embodiments, pis about 2, about 4, about 6 or about 8 Medicant units per antibody.

The average number of medicants per antibody in preparations of AMCsfrom conjugation reactions may be characterized by conventional meanssuch as UV/visible spectroscopy, mass spectrometry, ELISA assay, andHPLC. The quantitative distribution of AMCs in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous AMCs where p is a certain value from AMCwith other medicant loadings may be achieved by means such as reversephase HPLC or electrophoresis.

For some antibody medicant conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, an antibody may have only one or several cysteine thiolgroups, or may have only one or several sufficiently reactive thiolgroups through which a LU may be attached. In some embodiments, thecysteine thiol is a thiol group of a cysteine residue that forms aninterchain disulfide bond. In some embodiments, the cysteine thiol is athiol group of a cysteine residue that does not form an interchaindisulfide bond.

Typically, less than the theoretical maximums of medicant moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with theMedicant Linker compound intermediate or LU reagent. Only the mostreactive lysine groups may react with an amine-reactive LU reagent.Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a Medicant moiety via a LU.Most cysteine thiol residues in the antibodies exist as disulfidebridges and must be reduced with a reducing agent such as dithiothreitol(DTT). The antibody may be subjected to denaturing conditions to revealreactive nucleophilic groups such as lysine or cysteine. The loading(medicant/antibody ratio) of an AMC may be controlled in severaldifferent manners, including: (i) limiting the molar excess of MedicantLinker compound intermediate or LU reagent relative to antibody, (ii)limiting the conjugation reaction time or temperature, and (iii) partialor limiting reductive conditions for cysteine thiol modification.

Where more than one nucleophilic group reacts with a Medicant Linkercompound intermediate, or LU reagent followed by Medicant moietyreagent, then the resulting product is a mixture of Affinity MedicantLinker Conjugates (e.g., AMCs) with a distribution of one or moreMedicant moieties per Affinity unit (e.g., an antibody). The averagenumber of medicants per Affinity unit (e.g., antibody) may be calculatedfrom the mixture by, for example, dual enzyme linked immune serum assay(ELISA) antibody assay, specific for antibody and specific for themedicant. Individual Affinity Medicant Linker Conjugate molecules may beidentified in the mixture by mass spectroscopy, and separated by highperformance liquid chromatography (HPLC), e.g., hydrophobic interactionchromatography. Thus, a homogeneous conjugate with a single loadingvalue may be isolated from the conjugation mixture by electrophoresis orchromatography.

A “Linker Unit” (LU) is a bifunctional compound which can be used tolink a Medicant unit and/or an Affinity unit to form an AffinityMedicant Linker conjugate. Such conjugates are useful, for example, inthe formation of immuno conjugates directed against tumor associatedantigens. Such conjugates allow the selective delivery of cytotoxicdrugs to tumor cells. A LU includes a traditional linker, a4-fluorosulfonyl benzoyl (4-FSB) linker, a 3-fluorosulfonyl benzoyl(3-FSB) linker a 2-fluorosulfonyl benzoyl (2-FSB) linker, a maleimide(Mal1) linker, an azlactone linker and a bridging amino acid.

A traditional linker is a linker as defined in Table I through Table VI,where the reagent column identifies various traditional linkers. AStretcher Unit includes two or more Linker Units.

A bridging amino acid means —NH—C(R′)H—CO— or —N(R″)—C(R′)H—CO—including glycine, L-alanine, L-serine, L-threonine, L-cysteine,L-valine, L-leucine, L-isoleucine, L-methionine, L-proline,L-phenylalanine, L-tyrosine, L-tryptophan, L-aspartic acid, L-glutamicacid, L-apsparagine, L-glutamine, L-histidine, L-lysine, L-arginine,L-homocysteine, L-selenocysteine, L-pyrrolysine, L-carnitine,L-hypusine, 2-aminoisobutyric acid, dehydroalanine, L-gamma-aminobutyricacid, L-ornithine, L-citrulline, L-α-Amino-n-butyric acid, L-Norvaline,L-Norleucine, L-Pipecolic acid, L-Alloisoleucine, L-α,β-diaminopropionicacid, L-α,γ-diaminobutyric acid, L-Allothreonine, L-α-Amino-n-heptanoicacid, L-Homoserine, β-Amino-n-butyric acid, β-Aminoisobutyric acid,γ-Aminobutyric acid, L-isovaline, L-Sarcosine, N-ethyl glycine, N-propylglycine, N-isopropyl glycine, L-N-methyl alanine, L-N-ethyl alanine,N-methyl β-alanine, N-ethyl β-alanine, Isoserine,L-α-hydroxy-γ-aminobutyric acid, L-diaminopimelic acid, cystathione,L-aminoisobutyric acid, dehydroalanine, delta-aminolevulinic acid,4-aminobenzoic acid, L-Hydroxyproline, Formylmethioinine, L-lanthionine,djenkolic acid, L-Pyroglutamic acid, Hypusine, L-carboxyglutamic acid,penicillamin, L-thialysine, quisqualic acid, L-canavine,L-azetidine-2-carboxylic acid, D-alanine, D-serine, D-threonine,D-cysteine, D-valine, D-leucine, D-isoleucine, D-methionine, D-proline,D-phenylalanine, D-tyrosine, D-tryptophan, D-aspartic acid, D-glutamicacid, D-apsparagine, D-glutamine, D-histidine, D-lysine, D-arginine,D-homocysteine, D-selenocysteine, D-pyrrolysine, D-carnitine,D-hypusine, D-gamma-aminobutyric acid, D-ornithine, D-citrulline,D-α-Amino-n-butyric acid, D-Norvaline, D-Norleucine, D-Pipecolic acid,D-Alloisoleucine, D-α,β-diaminopropionic acid, D-α,γ-diaminobutyricacid, D-Allothreonine, D-α-Amino-n-heptanoic acid, D-Homoserine,D-isovaline, D-Sarcosine, D-N-methyl alanine, D-N-ethyl alanine,D-α-hydroxy-γ-aminobutyric acid, D-diaminopimelic acid,D-aminoisobutyric acid, D-Hydroxyproline, D-lanthionine, D-Pyroglutamicacid, D-carboxyglutamic acid, D-thialysine, quisqualic acid, D-canavine,D-azetidine-2-carboxylic acid. A ‘modified bridging amino acid’ means abridging amino acid with R′ including a hydroxyl group that has beenesterified, a bridging amino acid with R′ including a sulphur atom wherethe sulphur atom has been reacted with an alkyl or other organic groupand/or a bridging amino acid with R′ including a primary amino groupthat has been converted into a secondary or tertiary amino group.

In one embodiment, the LU of the Medicant Linker compound and AffinityMedicant Linker conjugate has the formula: —W_(w)-A_(a) wherein -A- is aStretcher Unit; a is 1 or 2; each —W— is independently an Amino Acidunit; w is independently an integer ranging from 1 to 20. In theAffinity Medicant Linker conjugate, the LU serves to attach the Medicantmoiety and the AM.

The Affinity Moiety (AM) includes within its scope an Affinity Unit (AU)that specifically binds or reactively associates or complexes with areceptor, antigen or other receptive moiety associated with a giventarget-cell population. An AU is a molecule that binds to, complexeswith, or reacts with a receptor, antigen or other receptive moiety of acell population sought to be therapeutically or otherwise biologicallymodified. In one aspect, the AM acts to deliver the Medicant unit to theparticular target cell population with which the AM interacts. Such AM'sinclude, but are not limited to, proteins, polypeptides and peptides andinclude, antibodies, binding proteins, smaller molecular weightproteins, polypeptides, peptides, lectins, glycoproteins, non-peptides,vitamins, nutrient-transport molecules (such as, but not limited to,transferrin), or any other cell binding molecule or substance.

In an embodiment of the invention, an AM can form a bond to a StretcherUnit. In an alternative embodiment of the invention, an AM can form abond to the Stretcher Unit of the LU via a heteroatom of the AM.Heteroatoms that may be present on an AM include sulfur (in oneembodiment, from a sulfhydryl group of an AM), oxygen (in oneembodiment, from a carbonyl, carboxyl or hydroxyl group of an AM) andnitrogen (in one embodiment, from a primary or secondary amino group ofan AM). These hetero atoms can be present on the AM in the AM's naturalstate, for example a naturally-occurring antibody, or can be introducedinto the AM via chemical modification.

In one embodiment, an AM unit has a sulfhydryl group and the AM bonds tothe LU via the sulfhydryl group's sulfur atom. In another embodiment,the AM has lysine residues that can react with activated esters (suchesters include, but are not limited to, N-hydroxysuccinimide,pentafluorophenyl, and p-nitrophenyl esters) of the Stretcher Unit ofthe AM and thus form an amide bond consisting of the primary nitrogenatom of the AM and the carboxyl group of the AM. In yet another aspect,the AM has one or more lysine residues that can be chemically modifiedto introduce one or more sulfhydryl groups. The AM bonds to the LU viathe sulfhydryl group's sulfur atom. The reagents that can be used tomodify lysines include, but are not limited to, N-succinimidylS-acetylthioacetate (SATA) and 2-Iminothiolane hydrochloride (Traut'sReagent).

In another embodiment, the AM can have one or more carbohydrate groupsthat can be chemically modified to have one or more sulfhydryl groups.The AM bonds to the LU (or a Stretcher Unit) via the sulfhydryl group'ssulfur atom. In yet another embodiment, the AM can have one or morecarbohydrate groups that can be oxidized to provide an aldehyde (—CHO)group. The corresponding aldehyde can form a bond with a reactive siteon a Stretcher Unit. Reactive sites on a Stretcher Unit that can reactwith a carbonyl group on an AM include, but are not limited to,hydrazine and hydroxylamine.

Useful non-immunoreactive protein, polypeptide, or peptide affinitymoieties include, but are not limited to, transferrin, epidermal growthfactors (“EGF”), bombesin, gastrin, gastrin-releasing peptide,platelet-derived growth factor, IL-2, IL-6, transforming growth factors(“TOP”), such as TGF-.alpha. and TGF-.beta., vaccinia growth factor(“VGF”), insulin and insulin-like growth factors I and II, somatostatin,lectins and apoprotein from low density lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Useful monoclonalantibodies are homogeneous populations of antibodies to a particularantigenic determinant (e.g., a cancer cell antigen, a viral antigen, amicrobial antigen, a protein, a peptide, a carbohydrate, a chemical,nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to anantigen-of-interest can be prepared by using any technique known in theart which provides for the production of antibody molecules bycontinuous cell lines in culture.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric monoclonal antibodies. Human monoclonalantibodies may be made by any of numerous techniques known in the art.

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art and are discussed infra.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to target cells (e.g.,cancer cell antigens, viral antigens, or microbial antigens) or otherantibodies that bind to tumor cells or matrix. In this regard,“functionally active” means that the fragment, derivative or analog isable to elicit anti-anti-idiotype antibodies that recognize the sameantigen that the antibody from which the fragment, derivative or analogis derived recognized. Specifically, in an exemplary embodiment theantigenicity of the idiotype of the immunoglobulin molecule can beenhanced by deletion of framework and CDR sequences that are C-terminalto the CDR sequence that specifically recognizes the antigen. Todetermine which CDR sequences bind the antigen, synthetic peptidescontaining the CDR sequences can be used in binding assays with theantigen by any binding assay method known in the art (e.g., the BIA coreassay).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, Fab fragments, Fvs, single chainantibodies, diabodies, triabodies, tetrabodies, scFv, scFv-FV, or anyother molecule with the same specificity as the antibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as for example,those having a variable region derived from a murine monoclonal andhuman immunoglobulin constant regions. Humanized antibodies are antibodymolecules from non-human species having one or more complementaritydetermining regions (CDRs) from the non-human species and a frameworkregion from a human immunoglobulin molecule. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. Other human antibodies can be obtainedcommercially from, for example, Abgenix, Inc. (now Amgen, Freemont,Calif.) and Medarex (Princeton, N.J.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. Human antibodies can also be producedusing various techniques known in the art, including phage displaylibraries.

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not from an antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation,derivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

Antibodies can have modifications (e.g., substitutions, deletions oradditions) in amino acid residues that interact with Fc receptors. Inparticular, antibodies can have modifications in amino acid residuesidentified as involved in the interaction between the anti-Fc domain andthe FcRn receptor.

Antibodies immunospecific for a cancer cell antigen can be obtainedcommercially or produced by any method known to one of skill in the artsuch as, e.g., chemical synthesis or recombinant expression techniques.The nucleotide sequence encoding antibodies immunospecific for a cancercell antigen can be obtained, e.g., from the GenBank database or adatabase like it, literature publications, or by routine cloning andsequencing.

In a specific embodiment, known antibodies for the treatment of cancercan be used. Antibodies immunospecific for a cancer cell antigen can beobtained commercially or produced by any method known to one of skill inthe art such as, e.g., recombinant expression techniques. The nucleotidesequence encoding antibodies immunospecific for a cancer cell antigencan be obtained, e.g., from the GenBank database or a database like it,the literature publications, or by routine cloning and sequencing.Examples of antibodies available for the treatment of cancer include,but are not limited to, RITUXAN® (rituximab; Genentech) which is achimeric anti-CD20 monoclonal antibody for the treatment of patientswith non-Hodgkin's lymphoma; OVAREX which is a murine antibody for thetreatment of ovarian cancer; PANOREX (Glaxo Wellcome, N.C.) which is amurine IgG_(2a) antibody for the treatment of colorectal cancer;Cetuximab ERBITUX (Imclone Systems Inc., NY) which is an anti-EGFR IgGchimeric antibody for the treatment of epidermal growth factor positivecancers, such as head and neck cancer; Vitaxin (MedImmune, Inc., MD)which is a humanized antibody for the treatment of sarcoma; CAMPATH I/H(Leukosite, MA) which is a humanized IgG₁ antibody for the treatment ofchronic lymphocytic leukemia (CLL); SMART MI95 (Protein Design Labs,Inc., CA) and SGN-33 (Seattle Genetics, Inc., WA) which is a humanizedanti-CD33 IgG antibody for the treatment of acute myeloid leukemia(AML); LYMPHOCIDE (Immunomedics, Inc., NJ) which is a humanizedanti-CD22 IgG antibody for the treatment of non-Hodgkin's lymphoma;SMART ID10 (Protein Design Labs, Inc., CA) which is a humanizedanti-HLA-DR antibody for the treatment of non-Hodgkin's lymphoma;ONCOLYM (Techniclone, Inc., CA) which is a radiolabeled murineanti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma;ALLOMUNE (BioTransplant, CA) which is a humanized anti-CD2 mAb for thetreatment of Hodgkin's Disease or non-Hodgkin's lymphoma; AVASTIN(Genentech, Inc., CA) which is an anti-VEGF humanized antibody for thetreatment of lung and colorectal cancers; Epratuzamab (Immunomedics,Inc., NJ and Amgen, Calif.) which is an anti-CD22 antibody for thetreatment of non-Hodgkin's lymphoma; and CEACIDE (Immunomedics, N.J.)which is a humanized anti-CEA antibody for the treatment of colorectalcancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens (where exemplarycancers that can be treated with the antibody are in parentheses): Alk(adrenocarcinomas) (SEQ. ID. 103), CAl25 (ovarian) (SEQ. ID. 104),CA15-3 (carcinomas) (SEQ. ID. 105), CA19-9 (carcinomas), L6 (carcinomas)(SEQ. ID. 107), Lewis Y (carcinomas) (SEQ. ID. 108), Lewis X(carcinomas) (SEQ. ID. 109), alpha fetoprotein (carcinomas) (SEQ. ID.110), CA 242 (colorectal), placental alkaline phosphatase (carcinomas)(SEQ. ID. 112), prostate specific antigen (prostate) (SEQ. ID. 113),prostate specific membrane antigen (prostate) (SEQ. ID. 114), prostaticacid phosphatase (prostate) (SEQ. ID. 115), epidermal growth factor(carcinomas) (SEQ. ID. 116), MAGE-1 (carcinomas) (SEQ. ID. 117), MAGE-2(carcinomas) (SEQ. ID. 118), MAGE-3 (carcinomas) (SEQ. ID. 119), MAGE-4(carcinomas) (SEQ. ID. 120), anti-transferrin receptor (carcinomas)(SEQ. ID. 121), p97 (melanoma) (SEQ. ID. 122), MUC1 (breast cancer)(SEQ. ID. 123), CEA (colorectal) (SEQ. ID. 124), gp100 (melanoma) (SEQ.ID. 125), MART-1 (melanoma) (SEQ. ID. 126), IL-2 receptor (T-cellleukemia and lymphomas) (SEQ. ID. 127), CD2 (buccal mucosa) (SEQ. ID.128), CD20 (non-Hodgkin's lymphoma) (SEQ. ID. 129), CD52 (leukemia)(SEQ. ID. 130), CD33 (leukemia) (SEQ. ID. 131), CD22 (lymphoma) (SEQ.ID. 132), beta human chorionic gonadotropin (carcinoma) (SEQ. ID. 133),CD38 (multiple myeloma) (SEQ. ID. 134), CD40 (lymphoma) (SEQ. ID. 135),CD80 (colorectal) (SEQ. ID. 136), CD86 (colorectal) (SEQ. ID. 137),mucin (carcinomas) (SEQ. ID. 138), P21 (carcinomas) (SEQ. ID. 139), MPG(melanoma) (SEQ. ID. 140), Neu oncogene product (carcinomas) (SEQ. ID.141) and STEAP-1 (prostate) (SEQ. ID. 142).

Compositions and Methods of Administration. In other embodiments,described is a pharmaceutical composition including an effective amountof an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound and a pharmaceutically acceptable carrier or vehicle. Thecompositions are suitable for veterinary or human administration.

The present pharmaceutical compositions can be in any form that allowsfor the composition to be administered to a patient. For example, thecomposition can be in the form of a solid or liquid. Typical routes ofadministration include, without limitation, parenteral, ocular andintra-tumor. Parenteral administration includes subcutaneous injections,intravenous, intramuscular or intrasternal injection or infusiontechniques. In one aspect, the compositions are administeredparenterally. In a specific embodiment, the compositions areadministered intravenously.

Pharmaceutical compositions can be formulated so as to allow an AffinityMedicant Linker conjugate and/or a Medicant Linker compound to bebioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit, and a container of anAffinity Medicant Linker conjugate and/or a Medicant Linker compound inliquid form can hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the Affinity Medicant Linker conjugateand/or a Medicant Linker compound, the manner of administration, and thecomposition employed.

The pharmaceutically acceptable carrier or vehicle can be solid orparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) can be liquid. In addition, the carrier(s)can be particulate.

The composition can be in the form of a liquid, e.g., a solution,emulsion or suspension. In a composition for administration byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates,phosphates or amino acids and agents for the adjustment of tonicity suchas sodium chloride or dextrose. A parenteral composition can be enclosedin ampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the Affinity Medicant Linker conjugate and/or a MedicantLinker compound that is effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays can optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in thecompositions will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.

The compositions comprise an effective amount of an Affinity MedicantLinker conjugate and/or a Medicant Linker compound such that a suitabledosage will be obtained. Typically, this amount is at least about 0.01%of an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound by weight of the composition. In an exemplary embodiment,pharmaceutical compositions are prepared so that a parenteral dosageunit contains from about 0.01% to about 2% by weight of the AffinityMedicant Linker conjugate and/or a Medicant Linker compound.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of an Affinity Medicant Linker conjugate and/or aMedicant Linker compound per kg of the patient's body weight. In oneaspect, the composition can include from about 1 to about 100 mg of anAffinity Medicant Linker conjugate and/or a Medicant Linker compound perkg of the patient's body weight. In another aspect, the amountadministered will be in the range from about 0.1 to about 25 mg/kg ofbody weight of the Affinity Medicant Linker conjugate and/or a MedicantLinker compound.

Prior art ADC's such as Kadcyla or Adcetris deliver a dose of theassociated toxin (auristatins MMAE or emtansine DM-1) that is three ormore times the lethal dose (for that toxin) which results in severesystemic (or non-target) toxicity. In contrast, Illudin and AcylfulveneADC's (such as analog 189 (FIG. 20HF, analog 190 (FIG. 20HG), analog 217(FIG. 20IH), analog 218 (FIG. 20II), analog 219 (FIG. 20IJ), analog 222(FIG. 20IM), or analog 316 (FIG. 20MC) deliver less than one third(i.e., <⅓) of a lethal dose, minimizing the risk and severity ofsystemic toxicity. Illudins and Acylfulvenes are true cytotoxic agentswhereas other toxic agents used in prior art ADC's (e.g.,pyrrolobenzodiazepines, maytansines, fumagillols, dolstatins,auristatins, enadiynes, halichondrins, and tubulysins) are onlycytostatic. See Table XIII (the NCI-DTP 60 cell line table). Hence,other payloads, such as those used in Herceptin, Adcetris or Rituxinonly stall tumor cell growth and do not actually kill the tumor cells.Other payloads (e.g., pyrrolobenzodiazepines, maytansines, fumagillols,dolstatins, auristatins, enadiynes, halichondrins, and tubulysins) arenot active against multidrug phenotypes, notably the MDR1/GP170 andMRP/GP180 transport mechanisms (see Table XIV). Illudins andAcylfulvenes show the excellent effect of remaining active against allMDR phenotypes known regardless of the mechanism of resistance (seeTable XIV). Hence, if tumor cells have already developed multi-drugresistance to a prior art ADC with a prior art toxin, or developmulti-drug resistance during the administration of a course of the priorart ADC with a prior art toxin, then the ADC will have no efficacy. Incontrast, ADCs developed with Illudins, Syn-illudins, or Acylfulveneshave the advantageous effect that they will continue to kill cancercells.

Generally, the dosage of an Affinity Medicant Linker conjugate and/or aMedicant Linker compound administered to a patient is typically about0.01 mg/kg to about 20 mg/kg of the patient's body weight. In oneaspect, the dosage administered to a patient is between about 0.01 mg/kgto about 10 mg/kg of the patient's body weight. In another aspect, thedosage administered to a patient is between about 0.1 mg/kg and about 10mg/kg of the patient's body weight. In yet another aspect, the dosageadministered to a patient is between about 0.1 mg/kg and about 5 mg/kgof the patient's body weight. In yet another aspect the dosageadministered is between about 0.1 mg/kg to about 3 mg/kg of thepatient's body weight. In yet another aspect, the dosage administered isbetween about 1 mg/kg to about 3 mg/kg of the patient's body weight.

The Affinity Medicant Linker conjugate and/or a Medicant Linker compoundcan be administered by any convenient route, for example by infusion orbolus injection. Administration can be systemic or local. Variousdelivery systems are known, e.g., encapsulation in liposomes,micro-particles, microcapsules, capsules, etc., and can be used toadminister an Affinity Medicant Linker conjugate and/or a MedicantLinker compound. In certain embodiments, more than one Affinity MedicantLinker conjugate and/or a Medicant Linker compound is administered to apatient.

In specific embodiments, it can be desirable to administer one or moreAffinity Medicant Linker conjugates and/or a Medicant Linker compoundlocally to the area in need of treatment. This can be achieved, forexample, and not by way of limitation, by local infusion during surgery;topical application, e.g., in conjunction with a wound dressing aftersurgery; by injection; by means of a catheter; or by means of animplant, the implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of a cancer, tumor or neoplastic or pre-neoplastictissue. In another embodiment, administration can be by direct injectionat the site (or former site) of a manifestation of an autoimmunedisease.

In yet another embodiment, the Affinity Medicant Linker conjugate and/ora Medicant Linker compound can be delivered in a controlled releasesystem, such as but not limited to, a pump or various polymericmaterials can be used. In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the Linker Affinityconjugate and/or a Medicant Linker compound, e.g., the liver, thusrequiring only a fraction of the systemic dose.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich an Affinity Medicant Linker conjugate and/or a Medicant Linkercompound is administered. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin. The carriers can be saline, and the like. Inaddition, auxiliary, stabilizing and other agents can be used. In oneembodiment, when administered to a patient, the Affinity Medicant Linkerconjugate and/or the Medicant Linker compound and pharmaceuticallyacceptable carriers are sterile. Water is an exemplary carrier when theAffinity Medicant Linker conjugate and/or a Medicant Linker compound areadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. The present compositions, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents.

The present compositions can take the form of solutions, pellets,powders, sustained-release formulations, or any other form suitable foruse.

In an embodiment, the Affinity Medicant Linker conjugates and/orMedicant Linker compounds are formulated in accordance with routineprocedures as a pharmaceutical composition adapted for intravenousadministration to animals, particularly human beings. Typically, thecarriers or vehicles for intravenous administration are sterile isotonicaqueous buffer solutions. Where necessary, the compositions can alsoinclude a solubilizing agent. Compositions for intravenousadministration can optionally comprise a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where an AffinityMedicant Linker conjugate and/or Medicant Linker compound is to beadministered by infusion, it can be dispensed, for example, with aninfusion bottle containing sterile pharmaceutical grade water or saline.Where the Affinity Medicant Linker conjugate and/or Medicant Linkercompound is administered by injection, an ampoule of sterile water forinjection or saline can be provided so that the ingredients can be mixedprior to administration.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

Whether in solid or liquid form, the present compositions can include apharmacological agent used in the treatment of cancer, an autoimmunedisease or an infectious disease.

Treatment of Cancer. The Affinity Medicant Linker conjugates andMedicant Linker compounds are useful for inhibiting the multiplicationof a tumor cell or cancer cell, causing apoptosis in a tumor or cancercell, or for treating cancer in a patient. The Affinity Medicant Linkerconjugates and/or Medicant Linker compounds can be used accordingly in avariety of settings for the treatment of animal cancers. The AffinityMedicant Linker Conjugates can be used to deliver a Medicant or Medicantunit to a tumor cell or cancer cell. Without being bound by theory, inone embodiment, the AM of an Affinity Medicant Linker conjugate binds toor associates with a cancer-cell or a tumor-cell-associated antigen, andthe Affinity Medicant Linker conjugate can be taken up (internalized)inside a tumor cell or cancer cell through receptor-mediated endocytosisor other internalization mechanism. The antigen can be attached to atumor cell or cancer cell or can be an extracellular matrix proteinassociated with the tumor cell or cancer cell. Once inside the cell, oneor more specific peptide sequences within or at the Medicant unit'sproximal end of the LU are hydrolytically cleaved by one or more tumorcell or cancer cell-associated proteases, resulting in release of theMedicant unit. The released Medicant unit is then free to migrate withinthe cell and induce cytotoxic or cytostatic activities. The AffinityMedicant Linker conjugate also can be cleaved by an intracellularprotease to release the Medicant moiety. In an alternative embodiment,the Medicant or Medicant unit is cleaved from the Affinity MedicantLinker conjugate outside the tumor cell or cancer cell, and the Medicantor Medicant unit subsequently penetrates the cell.

The Affinity Medicant Linker conjugates provide conjugation-specifictumor or cancer medicant targeting, thus reducing general toxicity ofthe Medicant. The LUs stabilize the Affinity Medicant Conjugates inblood, yet are cleavable by tumor-specific proteases within the cell,liberating a Medicant unit.

In one embodiment, the AM binds to the tumor cell or cancer cell. Inanother embodiment, the AM binds to a tumor cell or cancer cell antigenwhich is on the surface of the tumor cell or cancer cell. In anotherembodiment, the AM binds to a tumor cell or cancer cell antigen which isan extracellular matrix protein associated with the tumor cell or cancercell.

The specificity of the AM for a particular tumor cell or cancer cell canbe important for determining those tumors or cancers that are mosteffectively treated. For example, an Affinity Medicant Linker conjugateand/or Medicant Linker compound having a BR96 AM can be useful fortreating antigen positive carcinomas including those of the lung,breast, colon, ovaries, and pancreas. Affinity Medicant Linkerconjugates having an anti-CD30 or an anti-CD70 binding affinity moietycan be useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with an AffinityMedicant Linker conjugate and/or a Medicant Linker compound include, butare not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, retinoblastoma blood-borne cancers,including but not limited to: acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CML”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma acute and chronic leukemias:lymphoblastic, myelogenous, lymphocytic, myelocytic leukemias Lymphomas:Hodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma,Waldenstrom's macroglobulinemia, Heavy chain disease, Polycythemia vera.

Multi-Modality Therapy for Cancer. Cancers, including, but not limitedto, a tumor, metastasis, or other disease or disorder characterized byuncontrolled cell growth, can be treated or inhibited by administrationof an Affinity Medicant Linker conjugate or Medicant Linker compound.

In other embodiments, methods for treating cancer are provided,including administering to a patient in need thereof an effective amountof an Affinity Medicant Linker conjugate and a chemotherapeutic agent.In one embodiment the chemotherapeutic agent is that with whichtreatment of the cancer has not been found to be refractory. In anotherembodiment, the chemotherapeutic agent is that with which the treatmentof cancer has been found to be refractory. The Affinity Medicant Linkerconjugates can be administered to a patient that has also undergonesurgery as treatment for the cancer.

In some embodiments, the patient also receives an additional treatment,such as radiation therapy. In a specific embodiment, the AffinityMedicant Linker conjugate is administered concurrently with thechemotherapeutic agent or with radiation therapy. In another specificembodiment, the chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of an AffinityMedicant Linker conjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents, such a standardof care chemotherapeutic agent(s), can be administered.

Additionally, methods of treatment of cancer with an Affinity MedicantLinker conjugate and/or a Medicant Linker compound are provided as analternative to chemotherapy or radiation therapy where the chemotherapyor the radiation therapy has proven or can prove too toxic, e.g.,results in unacceptable or unbearable side effects, for the subjectbeing treated. The patient being treated can, optionally, be treatedwith another cancer treatment such as surgery, radiation therapy orchemotherapy, depending on which treatment is found to be acceptable orbearable.

The Affinity Medicant Linker (AML) conjugates and/or Medicant Linker(ML) compounds can also be used in an in vitro or ex vivo fashion, suchas for the treatment of certain cancers, including, but not limited toleukemia and lymphomas, such treatment involving autologous stem celltransplants. This can involve a multi-step process in which the animal'sautologous hematopoietic stein cells are harvested and purged of allcancer cells, the animal's remaining bone-marrow cell population is theneradicated via the administration of a high dose of an AML conjugatesand/or ML compound with or without accompanying high dose radiationtherapy, and the stem cell graft is infused back into the animal.Supportive care is then provided while bone marrow function is restoredand the patient recovers.

Treatment of Autoimmune Diseases. The Affinity Medicant Linkerconjugates and Medicant Linker compounds are useful for killing orinhibiting the replication of a cell that produces an autoimmune diseaseor for treating an autoimmune disease. The Affinity Medicant Linkerconjugates and Medicant Linker compounds can be used accordingly in avariety of settings for the treatment of an autoimmune disease in apatient. The Affinity Medicant Linker conjugates can be used to delivera Medicant unit to a target cell. Without being bound by theory, in oneembodiment, the Affinity Medicant Linker conjugate associates with anantigen on the surface of a target cell, and the Affinity MedicantLinker conjugate is then taken up inside a target-cell throughreceptor-mediated endocytosis. Once inside the cell, one or morespecific peptide sequences within and/or Medicant unit proximal to theLU are enzymatically or hydrolytically cleaved, resulting in release ofthe Medicant or Medicant unit. The released Medicant or Medicant unit isthen free to migrate in the cytosol and induce cytotoxic or cytostaticactivities. The Affinity Medicant Linker conjugate also can be cleavedby an intracellular protease to release the Medicant or Medicant moiety.In an alternative embodiment, the Medicant is cleaved from the AffinityMedicant Linker conjugate outside the target cell, and the Medicant orMedicant unit subsequently penetrates the cell.

In an embodiment of the present invention, the AM binds to an autoimmuneantigen. In one aspect, the antigen is on the surface of a cell involvedin an autoimmune condition. In another embodiment, the AM binds to anautoimmune antigen which is on the surface of a cell. In one embodiment,the AM binds to activated lymphocytes that are associated with theautoimmune disease state.

In a further embodiment, the Affinity Medicant Linker conjugate orMedicant Linker compound kills or inhibit the multiplication of cellsthat produce an autoimmune antibody associated with a particularautoimmune disease.

In various embodiments of the present invention, the AML or AMconjugates can be used to treat particular types of autoimmune diseasesincluding, but not limited to, Th2 lymphocyte related disorders (e.g.,atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergicrhinitis, Omenn's syndrome, systemic sclerosis, and graft versus hostdisease); Thi lymphocyte-related disorders (e.g., rheumatoid arthritis,multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto'sthyroiditis, Grave's disease, primary biliary cirrhosis, Wegener'sgranulomatosis, and tuberculosis); activated B lymphocyte-relateddisorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes); and Active ChronicHepatitis, Addison's Disease, Allergic Alveolitis, Allergic Reaction,Allergic Rhinitis, Alport's Syndrome, Anaphlaxis, AnkylosingSpondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis,Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis,Behcet's Disease, Bird-Fancier's Lung, Bronchial Asthma, Caplan'sSyndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease, ChronicGlomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease,Congenital Rubella Infection, CREST Syndrome, Crohn's Disease,Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis, Discoid Lupus,Dressler's Syndrome, Eaton-Lambert Syndrome, Echovirus Infection,Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr VirusInfection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia,Wegener's Granulomatosis.

Multi-Medicant Therapy of Autoimmune Diseases. Methods for treating anautoimmune disease are also disclosed including administering to apatient in need thereof an effective amount of an Affinity MedicantLinker conjugates or Medicant Linker compound and another therapeuticagent known for the treatment of an autoimmune disease.

Treatment of Infectious Diseases. The Affinity Medicant Linkerconjugates and Medicant Linker compounds are useful for killing orinhibiting the multiplication of a cell that produces an infectiousdisease or for treating an infectious disease. The Affinity MedicantLinker conjugates and Medicant Linker compounds can be used accordinglyin a variety of settings for the treatment of an infectious disease in apatient. The Affinity Medicant Linker conjugates can be used to delivera Medicant unit to a target cell. In an embodiment of the presentinvention, the AM binds to the infectious disease cell.

In various embodiments of the present invention, the AML or AMconjugates kill or inhibit the multiplication of cells that produce aparticular infectious disease including, but not limited to, Diphtheria,Pertussis, Occult Bacteremia, Urinary Tract Infection, Gastroenteritis,Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy,Retropharyngeal Abcess, Impetigo, Ecthyma, Pneumonia, Endocarditis,Septic Arthritis, Pneumococca, Peritonitis, Bactermia, Meningitis, AcutePurulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis,Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax,Nocardiosis, Salmonella, Typhoid Fever, Dysentery, Conjunctivitis,Sinusitis, Brucellosis, Tullaremia, Cholera, Bubonic Plague, Tetanus,Necrotizing Enteritis, Actinomycosis, Mixed Anaerobic Infections,Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever,Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia,Trachoma, Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis, Coccidiodomycosis, Blastomycosis, Sporotrichosis,Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis,Mycetoma, Chromomycosis Rickettsial Diseases: Typhus, Rocky MountainSpotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses,Rickettsialpox, Q Fever, Bartonellosis Parasitic Diseases: Malaria,Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis,Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis,Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis,Cyclosporiasis, Microsporidiosis, Ascariasis, Whipworm Infection,Hookworm Infection, Threadworm Infection, Ocular Larva Migrans,Trichinosis, Guinea Worm Disease, Lymphatic Filariasis, Loiasis, RiverBlindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch,Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis,Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease,Alveolar Hydatid Disease Viral Diseases: Measles, Subacute sclerosingpanencephalitis, Common Cold, Mumps, Rubella, Roseola, Fifth Disease,Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis,Infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot-and-MouthDisease, Bornholm Disease, Genital Herpes, Genital Warts, AsepticMeningitis, Myocarditis, Pericarditis, Gastroenteritis, AcquiredImmunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV),Reye's Syndrome, Kawasaki Syndrome, Influenza, Bronchitis, Viral“Walking” Pneumonia, Acute Febrile Respiratory Disease, Acutepharyngoconjunctival fever, Epidemic keratoconjunctivitis, HerpesSimplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles,Cytomegalic Inclusion Disease, Rabies, Progressive MultifocalLeukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt-JakobDisease, Gerstmann-Straussler-Scheinker Disease, Tropical SpasticParaparesis, Western Equine Encephalitis, California Encephalitis, St.Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis,Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, MarburgVirus Infections, Ebola Virus Infections, Smallpox.

Synthesis of AMCs with SMCC and Sulfo-SMCC linkers. In an embodiment ofthe present invention, an affinity medicant conjugate (AMC) 1000 isformed between an AM 1100 and a medicant 1350 by reacting the medicant1350 with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate(SMCC) which forms an active intermediate that reacts with a sulfhydrylgroups on the AM 1100. In an embodiment of the present invention, theresulting AMC includes one or more molecules of the medicant 1350 boundto the AM 1100. In an embodiment of the present invention, the resultingAMC is not cleaved in the cytosol, but internalized and the AM 1100degraded by proteases in the cytosol until the medicant 1350 isreleased.

In an alternative embodiment of the present invention, an AMC 1000 isformed between an AM 1100 and a medicant 1350 by reacting the medicant1350 withsulfosuccinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene(Sulfo-SMCC) which forms an active intermediate that reacts with asulfhydryl groups on the AM 1100 to form a more water soluble AMC. In anembodiment of the present invention, the resulting AMC includes one ormore molecules of the medicant 1350 bound to the AM 1100. In anembodiment of the present invention, the resulting AMC is not cleaved inthe cytosol, but internalized and the AM 1100 degraded by proteases inthe cytosol until the medicant 1350 is released.

In an embodiment of the present invention, an AMC 1000 comprises an AM1100 bound to a medicant 1350 through an optional linker as illustratedin FIG. 1. In an embodiment of the present invention, an antibody 1110is bound to a linker 1200 which is bound to the medicant 1350. In anunexpected result, an AMC 1000 can retain both the receptor bindingactivity of the AM 1100 and the intracellular cytoactivity of themedicant 1350 in a single compound. In an embodiment of the presentinvention, an antibody 1110 is bound to a linker 1200 which is bound tothe medicant 1350. In an unexpected result, an antibody medicantconjugate can retain both the receptor binding activity of the antibody1110 and the intracellular cytoactivity of an acylfulvene in a singlecompound. Surprisingly, the antibody is capable of binding to apolypeptide receptor on cell populations thereby bringing theacylfulvene in contact with the cell population.

In an embodiment of the present invention, the medicant moiety is anacylfulvene moiety. An acylfulvene moiety includes irofulven derivatives(see structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I,FIG. 2L, FIG. 2M, FIG. 2P, FIG. 2S and FIG. 2U) and illudin derivatives(see structures shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J,FIG. 2K, FIG. 2N, FIG. 2O, FIG. 2Q, FIG. 2R, FIG. 2T, and FIG. 2V).

Amine Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes independently a carbon or a heteroatom containing nitrogen (N),oxygen (O) or sulphur (S); where R₆ denotes including —H, —CN, —CF₃, —O,—NH₂, —SO₃, —COOH—, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and where R₆ is NH₂ (an aminogroup) for an irofulvene derivative shown in FIG. 2A, FIG. 2C, FIG. 2F,FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudin derivative shown inFIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG.20.

Table IA shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene amino derivative shown in the first column ofTable IA is linked to the AM through the free sulfhydryl group of the AMusing the reagent identified in the second column of Table IA to formthe AMC.

FIG. 8A shows the structure of the analog 211 (FIG. 20IB) attached viathe amino group using the SMPT linking reagents. FIG. 8B shows thestructure of the analog 211 (FIG. 20IB) attached via the amino groupusing the SMCC linking reagent. FIG. 8C shows the structure of theanalog 211 (FIG. 20IB) attached via the amino group using the SIABlinking reagent.

Table IB shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM via aphotoactivatable group at the other terminus using the reagent. In anembodiment of the present invention, the acylfulvene amino derivativeshown in the first column of Table IB is linked to the AM to the AMthrough the photoactivatable group at the other terminus using thereagent identified in the second column of Table IB to form the AMC.

Table IC shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM through areactive amine group at the other terminus using the reagent. In anembodiment of the present invention, the acylfulvene amino derivativeshown in the first column of Table IC is linked to the AM through anamine reactive group using the reagent identified in the second columnof Table IC to form the AMC.

Table ID shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to the AM through analdehyde, carbonyl or carboxylate group at the other terminus using thereagent. In an embodiment of the present invention, the acylfulveneamino derivative shown in the first column of Table ID is linked to theAM through an aldehyde, carbonyl or carboxylate group at the otherterminus using the reagent identified in the second column of Table IDto form the AMC.

Carboxyl Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH and where R₆ is CO₂H (a carboxyl group) for an irofulvenederivative shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2Land FIG. 2M and illudin derivative shown in FIG. 2B, FIG. 2D, FIG. 2E,FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 20. R₅ is glycine or eitheran L or D amino acid including alanine, serine, threonine, cysteine,valine, leucine, isoleucine, methionine, proline, phenylalanine,tyrosine, tryptophan, aspartic acid, glutamic acid, apsparagine,glutamine, histidine, lysine, arginine, alpha-methyl glycine or2-dimethylglycine. R₅ can also comprise nonstandard amino acids toreduce nonspecific esterase activity present in blood and cellsincluding homocysteine, selenocysteine, pyrrolysine, carnitine,hypusine, lanthionine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, ornithine, citrulline, α-Amino-n-butyric acid,Norvaline, Norleucine, Pipecolic acid, Alloisoleucine,α,β-diaminopropionic acid, α,γ-diaminobutyric acid, Allothreonine,α-Amino-n-heptanoic acid, Homoserine, β-Amino-n-butyric acid,β-Aminoisobutyric acid, α-Aminobutyric acid, isovaline, Sarcosine,N-ethyl glycine, N-propyl glycine, N-isopropyl glycine, N-methylalanine, N-ethyl alanine, N-methyl β-alanine, N-ethyl β-alanine,Isoserine, α-hydroxy-γ-aminobutyric acid, diaminopimelic acid,cystathione, aminoisobutyric acid, dehydroalanine,delta-aminolevulinicacid, 4-aminobenzoic acid, Hydroxyproline, Formylmethioinine,lanthionine, djenkolic acid, Pyroglutamic acid, Hypusine,carboxyglutamic acid, penicillamin, thialysine, quisqualic acid,canavine, azetidine-2-carboxylic acid. FIG. 9A shows the structure ofthe analog 038 (FIG. 20BL) attached via the carboxyl group using the EDClinking reagent. FIG. 9B shows the structure of the analog 038 (FIG.20BL) attached via the carboxyl group using the CMC linking reagent.FIG. 9C shows the structure of the analog 038 (FIG. 20BL) attached viathe carboxyl group using the DCC linking reagent.

Table IIA shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker which can then be attached to a sulfhydrylreacting group of the AM. In an embodiment of the present invention, theacylfulvene carboxylate derivative shown in the first column of TableIIA is linked to the AM through the free sulfhydryl group of the AMusing the reagent identified in the second column of Table IIA to formthe AMC.

Table IIB shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM. In anembodiment of the present invention, the acylfulvene carboxylatederivative shown in the first column of Table IIB is linked to the AMthrough the photoactivatable reactive group using the reagent identifiedin the second column of Table IIB to form the AMC.

Table IIC shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains an amino reactivegroup which can attach to the AM. In an embodiment of the presentinvention, the acylfulvene carboxylate derivative shown in the firstcolumn of Table IIC is linked to the AM through the amino group usingthe reagent identified in the second column of Table IIC to form theAMC.

Azlactone Derivative. FIG. 10A shows the structure of the analog 038(FIG. 20BL) attached via the carboxyl group using DCC or DIC linkingreagents in the presence of glycine. FIG. 10B shows the structure of theanalog 038 (FIG. 20BL) attached via the carboxyl group using DCC or DIClinking reagents in the presence of alanine. FIG. 10C shows thestructure of the analog 106 attached via the carboxyl group using DCC orDIC linking reagents in the presence of valine.

Table IID shows acylfulvene carboxylate analogs which can be reacted toform acylfulvene azlactone derivatives where the azlactone reactivegroup can be used to attach to the AM. In an embodiment of the presentinvention, the acylfulvene derivative shown in the first column of TableIID is converted to the acylfulvene azlactone derivative (see FIG. 2P)using the reagent identified in the second column of Table IID to formthe AMC.

Carbonyl Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁and R₇ denote independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl inwhich incorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen(N) functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH and where R₆ is CO—R₇ (acarbonyl linking group) for an irofulvene derivative shown in FIG. 2A,FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2L and FIG. 2M and illudinderivative shown in FIG. 2B, FIG. 2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG.2K, FIG. 2N and FIG. 2O.

FIG. 11A shows the structure of the analog 124 (FIG. 20ET) attached viathe carbonyl group using the AMBH linking reagent. FIG. 11B shows thestructure of the analog 124 (FIG. 20ET) attached via the carbonyl groupusing the ABH linking reagent. FIG. 11C shows the structure of theanalog 201 (FIG. 20HR) attached via the M₂C₂H linking reagent.

Table IIIA shows acylfulvene carbonyl analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene carbonyl derivative shown in the first columnof Table IIIA is linked to the AM through the free sulfhydryl group ofthe AM using the reagent identified in the second column of Table IIIAto form the AMC.

Table IIIB shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent. In anembodiment of the present invention, the acylfulvene carbonyl derivativeshown in the first column of Table IIIB is linked to the AM through thephotoactivatable reactive group using the reagent identified in thesecond column of Table IIIB to form the AMC.

Table IIIC shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene carbonyl derivative shown in thefirst column of Table IIIC is linked to the AM through the amino groupusing the reagent identified in the second column of Table IIIC to formthe AMC.

Aldehyde Derivative. In an embodiment of the present invention, theirofulvene structures shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG.2I, FIG. 2L and FIG. 2M and illudin structures shown in FIG. 2B, FIG.2D, FIG. 2E, FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O, where R₁denotes substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH and where R₆ is HCO (an aldehyde group) for an irofulvenederivative shown in FIG. 2A, FIG. 2C, FIG. 2F, FIG. 2H, FIG. 2I, FIG. 2Land FIG. 2M and illudin derivative shown in FIG. 2B, FIG. 2D, FIG. 2E,FIG. 2G, FIG. 2J, FIG. 2K, FIG. 2N and FIG. 2O.

FIG. 12A shows the structure of the analog 010 (FIG. 20AJ) attached viathe aldehyde group using the PDPH linking reagent. FIG. 12B shows thestructure of the analog 010 (FIG. 20AJ) attached via the aldehyde groupusing the ABH linking reagent. FIG. 12C shows the structure of theanalog 011 (FIG. 20AK) attached via MPBH linking reagent.

Table IVA shows acylfulvene aldehyde analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene aldehyde derivative shown in the first columnof Table IVA is linked to the AM through the free sulfhydryl group ofthe AM using the reagent identified in the second column of Table IVA toform the AMC.

Table IVB shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent. In anembodiment of the present invention, the acylfulvene aldehyde derivativeshown in the first column of Table IVB is linked to the AM through thephotoactivatable reactive group using the reagent identified in thesecond column of Table IVB to form the AMC.

Table IVC shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene aldehyde derivative shown in thefirst column of Table IVC is linked to the AM through the amino groupusing the reagent identified in the second column of Table IVC to formthe AMC.

Alcohol Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ denotessubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH for an irofulven derivative (FIG. 2C), an illudin ringderivative (FIG. 2D) or an illudin alkyl derivative (FIG. 2E). FIG. 13Ashows the structure of the analog 009 (FIG. 20AI) attached via thealcohol group using the CDI linking reagent. FIG. 13B shows thestructure of the analog 009 (FIG. 20AI) attached via the alcohol groupusing the HSC linking reagent. FIG. 13C shows the structure of themedicant moiety Illudin M attached via the DSC linking reagent.

Table VA shows acylfulvene alcohol analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent. In an embodiment of the presentinvention, the acylfulvene alcohol derivative shown in the first columnof Table VA is linked to the AM through the free sulfhydryl group of theAM using the reagent identified in the second column of Table VA to formthe AMC.

Table VB shows acylfulvene alcohol analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent. In an embodiment ofthe present invention, the acylfulvene alcohol derivative shown in thefirst column of Table VB is linked to the AM through the amino groupusing the reagent identified in the second column of Table VB to formthe AMC.

Sulfhydryl Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ and R₆ denoteindependently substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, or CH₂OH, and R₂, R₃, R₄, R₅ denoteeither H, CH₃, or CH₂OH and R₇ is SH or SS— R₈ for an irofulvenderivative (FIG. 2C), an illudin ring derivative (FIG. 2D) or an illudinalkyl derivative (FIG. 2E). FIG. 14A shows the structure of the analog051 (FIG. 20BY) attached via the sulfhydryl group using SMCC linkingreagent. FIG. 14B shows the structure of the analog 051 (FIG. 20BY)attached via the sulfhydryl group using MPBH linking reagent. FIG. 14Cshows the structure of the analog 051 (FIG. 20BY) attached via thesulfhydryl group using PDPH linking reagent.

In an embodiment of the present invention, analog 051 (FIG. 20BY) can beattached to an AM by attaching a disulfide bridge at 6′ position, aterminal cysteine or n-acetylcysteine group. Analog 051 (FIG. 20BY) hasa free sulfhydryl group which can react with other sulfhydryl groups toproduce a disulfide bond or alternatively react with specificsulfhydryl-reacting groups such as malonic acid derivatives. The othersulfhydryl groups can be on a linker, where the free sulfhydryl groupwill react with sulfhydryl reactive groups on the linkers, e.g., malonicacid derivatives such as SMCC. Alternatively a medicant with a freesufhydryl can directly react with free sulfhydryl groups on an AM (suchas are present in cysteine residues).

Table VIA shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent (a reducing agent canbe used to reduce the disulfide and generate a sulfhydryl group). In anembodiment of the present invention, the acylfulvene sulfhydrylderivative shown in the first column of Table VIA is linked to the AMthrough the free amino group of the bi-functional linker using thereagent identified in the second column of Table VIA to form the AMC.

Table VIB shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a sulfhydrylreacting group which can attach to the AM using the reagent (a reducingagent can be used to reduce the disulfide and generate a sulfhydrylgroup). In an embodiment of the present invention, the acylfulvenesulfhydryl derivative shown in the first column of Table VIB is linkedto the AM through the free sulfhydryl group of the bi-functional linkerusing the reagent identified in the second column of Table VIB to formthe AMC.

Table VIC shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM using thereagent (a reducing agent can be used to reduce the disulfide andgenerate a sulfhydryl group). In an embodiment of the present invention,the acylfulvene sulfhydryl derivative shown in the first column of TableVIC is linked to the AM through the photoactivatable reactive group ofthe bi-functional linker using the reagent identified in the secondcolumn of Table VIC to form the AMC.

Table VID shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a carboxylatereactive group which can attach to the AM using the reagent (a reducingagent can be used to reduce the disulfide and generate a sulfhydrylgroup). In an embodiment of the present invention, the acylfulvenesulfhydryl derivative shown in the first column of Table VID is linkedto the AM through the carboxylate reactive group of the bi-functionallinker using the reagent identified in the second column of Table VID toform the AMC.

Halide Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2C, FIG. 2D, and FIG. 2E, R₁ denotessubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH and Xis a halogen for an irofulven derivative (FIG. 2C),an illudin ring derivative (FIG. 2D) or an illudin alkyl derivative(FIG. 2E).

In an embodiment of the present invention, the medicant moieties 4 (FIG.20AD), 5 (FIG. 20AE), 20 (FIG. 20AT), 53 (FIG. 20CA), 237 (FIG. 20JB)which contain halide groups can react in one of two ways. They willreact directly with free sulfhydryl groups present onantibodies/proteins (e.g., on cysteine residues) or they can react withsulfhydryl groups on linkers (e.g., such as malonic acid derivativessuch as SMCC). FIG. 21 shows analog 20 (FIG. 20AT) linked to DSP (FIG.21A), DTME (FIG. 21B) and SMPT (FIG. 21C).

Acyl Azide or Azide Derivative. In an embodiment of the presentinvention, the structures shown in FIG. 2F and FIG. 2G, R₁ denotesindependently substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote either H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃, OCH₂CH₃ for anirofulven derivative (FIG. 2F) or an illudin derivative (FIG. 2G).

In an embodiment of the present invention, the medicant moieties 193(FIG. 20HJ), 195 (FIG. 20HL), 299 (FIG. 20LL), 300 (FIG. 20LM), 307(FIG. 20LT) can be photoactivated, with UV radiation. The acyl azidesand phenylazides do not need linkers, forming a reactive nitrene groupthat reacts with primary amines on proteins. The only caveat is thereaction of the drug and protein must be carried out in the absence ofthiol reducing agents.

The azide must be on a ring system like a benzene or phenyl group, seeanalogs 193 (FIG. 20HL), 195 (FIG. 20HL), 300 (FIG. 20LM), 307 (FIG.20LT) and 309 (FIG. 20LV).

Epoxide Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2H, FIG. 2I, FIG. 2J and FIG. 2K, where R₁substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl in which incorporatedheteroatoms can be halogens (F, Cl, Br, I); nitrogen (N) functionalgroups including primary amines (—NH₂), secondary amines (—NH—),tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—), (—C(═N)R_(A)—), Azo(—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide (—C(═O)NR_(A)R_(B))or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S) functional groupsincluding thioethers (—S—), thiones (—C(═S)—, sulfoxides (—S(═O)—,sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))— or (—S(═O)(═NH)—,sulfhydryls (—SH), thiocyanate (—S—C(═N)—, isothiocyanate (—N═C(═S);oxygen (O) functional groups including hydroxyl (—OH), carbonyl(—C(═O)—), aldehyde (—C(═O)H, carboxylate (COOH), ethers (—O—), esters(—OC(═O)—), carbonate (—O(C═O)O—); and R₂, R₃, R₄, R₅ denote either H,CH₃, or CH₂OH, R₆ denotes independently halogen, —CN, —CF₃, —OH, —NH₂,—SO₂, —COOH, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and R₂,R₃, R₄, R₅ denote either H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃, OCH₂CH₃ and Xdenotes a heteroatom including oxygen (O), sulfur (S), and nitrogen (N)for irofulven derivatives (FIG. 2H and FIG. 2I) or illudin derivatives(FIG. 2J and FIG. 2K).

In an embodiment of the present invention, the medicant moieties 114(FIG. 20EJ) epoxides react with carboxyl groups, thiols, amines andhydroxyl groups. For example, analog 114 (FIG. 20EJ) can be linked toABH, BMPA, or PDPH.

EXAMPLE 1 Synthesis of Medicant 113

The Wittig reaction was performed on analog 10 (FIG. 20AJ). First 65 mgCH₃PPh₃Br (0.185 mmol) in anhydrous THF was cooled to −75° C. andstirred for 1 hour. Then 200 μL of n-butyl lithium (0.183 mmol) wasadded very slowly to the flask while maintaining temperature at −75° C.,and a yellow precipitate formed. It was stirred for another 1.5 hoursthen analog 10 (50 mg, 0.183 mmol) was slowly added while maintainingtemperature at −75° C., followed by stirring for 2.0 hours. The reactionwas quenched with ammonium chloride, extracted with CH₂Cl₂, washed withwater, NaHCO₃, and saline. Dried over Na₂SO₄ and concentrated. Theresidue was eluted through a column (10% ethyl acetate in hexane) togive analog 113 (FIG. 200) as a solid.

Acroyl Derivative. In an embodiment of the present invention, thestructures shown in FIG. 2L, FIG. 2M, and FIG. 2N and FIGS. 2O, R₁ andR₆ denote independently substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl in whichincorporated heteroatoms can be halogens (F, Cl, Br, I); nitrogen (N)functional groups including primary amines (—NH₂), secondary amines(—NH—), tertiary amines (—NR_(A)R_(B)), imine (—C(═N)H—),(—C(═N)R_(A)—), Azo (—N═N—), Cyanate (—C═N), isocyanate (—N═(C═O), amide(—C(═O)NR_(A)R_(B)) or (—C(═O)NR_(A)H) or (—C(═O)NH₂); sulfur (S)functional groups including thioethers (—S—), thiones (—C(═S)—,sulfoxides (—S(═O)—, sulfones (—S(═O)₂—), sulfoximes (—S(═O)(═NR_(A))—or (—S(═O)(═NH)—, sulfhydryls (—SH), thiocyanate (—S—C(═N)—,isothiocyanate (—N═C(═S); oxygen (O) functional groups includinghydroxyl (—OH), carbonyl (—C(═O)—), aldehyde (—C(═O)H, carboxylate(COOH), ethers (—O—), esters (—OC(═O)—), carbonate (—O(C═O)O—); and R₂,R₃, R₄, R₅ denote independently either H, CH₃, OH, OCH₃, CH₂OH, CH₂CH₃,OCH₂CH₃ and X denotes a heteroatom including oxygen (O), sulfur (S), andnitrogen (N) for irofulven derivatives (FIG. 2L and FIG. 2M) or illudinderivatives (FIG. 2N and FIG. 2O).

In an embodiment of the present invention, the medicant moieties 113(FIG. 20) react predominately with sulfhydryl groups. Acroyl derivativescan react in one of two ways. They will react directly with freesulfhydryl groups present on antibodies and proteins (e.g., on cysteineresidues) or they will react with sulfhydryl groups on linkers (e.g.,such as malonic acid derivatives such as SMCC).

Illudin1 linked to an Antibody. In various embodiments of the presentinvention, an AMC is made up of an antibody 1110 linked to an illudin1moiety 1301. Various embodiments of the invention, are directed to themethods for the preparation, use, and to pharmaceutical compositionscontaining an illudin1 moiety 1301 linked to an antibody 1110 to form anantibody medicant conjugate (AMC). In various embodiments the compoundsof the present invention, the AMC can have the general formula shown inFIG. 3A, where the antibody 1110 is bound to a linker 1200 which isbound to an illudin1 moiety 1301. In other various embodiments of thepresent invention, the compounds of the present AMC invention can havethe general formula shown in FIG. 3B, where a growth factor 1120 isbound to a linker 1200 which is bound to an illudin1 moiety 1301. Invarious embodiments the compounds of the present invention includestereoisomers, solvates, and pharmaceutically acceptable salts thereof,where the linker 1200 is as defined in Table X, and the illudin1 1301 isas defined below in Table XI.

Linker to bind Illudin to an Antibody. In an embodiment of the presentinvention, an antibody 1110 with a traditional linker 1240 to anilludin1 moiety 1301 binds to a receptor to which the antibody 1110 wasprepared and directs the illudin1 moiety 1301 to cell populationsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 bound with a traditional linker 1240 to an illudin1 moiety1301 acts as an AM for a receptor and directs the illudin1 moiety 1301to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, an antibody 1110 with a traditional linker 1240bound to an illudin1 moiety 1301 acts as an AM for a receptor anddirects the illudin1 moiety 1301 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, an antibody1110 with a traditional linker 1240 bound to an illudin1 moiety 1301acts as an AM for a receptor and directs the illudin1 moiety 1301 totumors containing cells over-expressing the receptor compared to nontumor cells.

Linker to bind Illudin to Growth Factor. In an embodiment of the presentinvention, an illudin1 moiety 1301 linked via a traditional linker 1240to a growth factor 1120 binds to the growth factor receptor and directthe illudin1 moiety 1301 to cell populations expressing the receptor. Inan embodiment of the present invention, a growth factor 1120 linked viaa traditional linker 1240 to an illudin1 moiety 1301 acts as an AM forthe growth factor receptor and directs the illudin1 moiety 1301 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a traditionallinker 1240 to an illudin1 moiety 1301 acts as an AM for the growthfactor receptor and directs the illudin1 moiety 1301 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a growth factor 1120 linked via a traditional linker1240 to an illudin1 moiety 1301 acts as an AM for the growth factorreceptor and directs the illudin1 moiety 1301 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Linker to bind Illudin to Steroid. Whereby Illudin S, Illudin M, or oneof analogs 001 through 316 (see FIG. 20) can be attached, eitherdirectly or with a linker, to a steroid which allows preferentialbinding to a cell overexpressing that particular receptor for thatsteroid and subsequent killing of the cell (see e.g., Table VII).

EXAMPLE 2 Synthesis of Medicant-Estrone 107

Analog 106 (see Example 13 and FIG. 20EB) (139 mg 0.384 mmol, 1 equiv.),DMAP (4 mg, 0.03 mmol, 0.08 equiv.) and estrone (104.4 mg, 0.384 mmol, 1equiv.) were dissolved in CH₂Cl₂ (14 mL) at 0° C. To this solution wasadded CH₂Cl₂ solution of DCC (460 μL, 1 M, 0.46 mmol, 1.2 equiv.)through a syringe. After 0.5 hours the solution was raised to roomtemperature. After 2 hours the mixture was filtered and the filtrate waswashed with dilute HCl (1.5%), saturated NaHCO₃ and brine in sequence.The organic phase was then dried and evaporated. The residue was elutedthrough a column (CH₂Cl₂/Methanol, 10:0.25) to give analog 107 (FIG.20EC) (100 mg, 42%) as semisolid. Analog 107 (FIG. 20EC) can besubsequently linked to estrone.

EXAMPLE 3 Preparation of Medicant-Estradiol 108

Analog 038 (FIG. 20BL) (58.5 mg, 0.2035 mmol), beta-estradiol (58.0 mg,0.2150 mmol) and DMAP (5 mg. 0.048 mmol) were dissolved in CH₂Cl₂ (5.6mL) at 0° C. To this solution was added CH₂Cl₂ solution of DCC (250 μL,1 M, 0.244 mmol), stirred for 30 minutes, allowed to warm to roomtemperature then stirred for 1.5 hours. The filtrate was washed withdilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. The organicphase was dried over Na₂SO₄, and evaporated. The residue was elutedthrough a column (100% CH₂Cl₂), fractions collected then eluted througha second column (CH₂Cl₂plus 0.5% methanol), to give analog 108 (FIG.20ED) (45 mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean±SD) for 108 (see FIG. 20). MCF7 over expressestrogen alpha-receptors. MCF7 cells are preferentially killed by 110(FIG. 20EG) the acylfulvene-estrone analog and to a lesser extent 108(FIG. 20ED) the acylfulvene-estradiol analog because estronepreferentially binds to alpha-receptor.

EXAMPLE 4 Preparation of Medicant-Estradiol 109

Analog 106 (FIG. 20EB) (54.5 mg, 0.15 mmol, 1 equiv.), β-estradiol (40.5mg, 0.15 mmol), and DMAP (1.8 mg, 0.015 mmol, 0.1 equiv.) were dissolvedin CH₂Cl₂ (5 mL) at 0° C. To this solution was added CH₂Cl₂ solution ofDCC (165 μL, 1 M, 0.165 mmol, 1.1 equiv.). The mixture was raised toroom temperature after 0.5 h. After another 2 h, the mixture wasfiltered. The filtrate was washed with dilute HCl (1.5%), saturatedNaHCO₃ and brine in sequence. The organic phase was dried andevaporated. The residue was eluted through a column (CH₂Cl₂/Methanol10:0.25) to give analog 109 (FIG. 20EE) (55 mg, 60%) as semisolid.

EXAMPLE 5 Preparation of Medicant-Estrone 110

Analog 038 (FIG. 20BL) (68 mg, 0.2365 mmol), estrone (68.0 mg, 0.2160mmol) and DMAP (5 mg. 0.048 mmol) were dissolved in CH₂Cl₂ (8.0 mL) at0° C. To this solution was added CH₂Cl₂ solution of DCC (300 μL, 1 M,0.283 mmol), stirred for 30 minutes, allowed to warm to room temperaturethen stirred for 0.5 hours. The filtrate was washed with dilute HCl(1.5%), saturated NaHCO₃ and brine in sequence. The organic phase wasdried over Na₂SO₄, and evaporated. The residue was eluted through acolumn (100% CH₂Cl₂), fractions collected then eluted through a secondcolumn (CH₂Cl₂plus 0.5% methanol), to give analog 110 (FIG. 20EF) (40mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean±SD) for 110 (FIG. 20EF). MCF7 cells over expressestrogen alpha-receptors. MCF7 cells are preferentially killed by theacylfulvene-estrone analog 110 (FIG. 20EF) and to a lesser extent by theacylfulvene-estradiol analog 108 (FIG. 20ED) because estronepreferentially binds to alpha-receptor. In contrast, illudin M killedboth ER negative and ER positive cells to the same extent. The data inTable VII demonstrates that compounds 108 (FIG. 20ED) and 110 (FIG.20EF) are preferentially cytotoxic to cells expressing large numbers ofestrogen receptors on their surface.

EXAMPLE 6 Preparation of Medicant-Testosterone 111

Analog 038 (FIG. 20BL) (52.5 mg, 0.182 mmol), testosterone (50.0 mg,0.173 mmol) and DMAP (5 mg. 0.048 mmol) were dissolved in CH₂Cl₂ (8.0mL) at 0° C. To this solution was added CH₂Cl₂ solution of DCC (250 μL,1 M), stirred for 30 minutes, allowed to warm to room temperature thenstirred for 2 hours. The filtrate was washed with dilute HCl (1.5%),saturated NaHCO₃ and brine in sequence. The organic phase was dried overNa₂SO₄, and evaporated. The residue was eluted through a column (100%CH₂Cl₂ plus 0.5% methanol), to give analog 111 (FIG. 20EG) (15 mg) as asolid.

EXAMPLE 7 Preparation of Medicant-Androsterone 112

Analog 038 (FIG. 20BL) (29 mg), androsterone (25.0 mg) and DMAP (5 mg.0.048 mmol) were dissolved in CH₂Cl₂ (5.0 mL) at 0° C. To this solutionwas added CH₂Cl₂ solution of DCC (150 μL, 1 M), stirred for 30 minutes,allowed to warm to room temperature then stirred for 2 hours. Thefiltrate was washed with dilute HCl (1.5%), saturated NaHCO₃ and brinein sequence. The organic phase was dried over Na₂SO₄, and evaporated.The residue was eluted through a column (2:3 ethyl acetate: hexane) togive analog 112 (FIG. 20EH) (15 mg) as a solid.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a traditional linker 1240 to a steroid 1140 bind to receptorsfor the steroid and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, asteroid 1140 linked via a traditional linker 1240 to an illudin2 moiety1302 acts as an AM for the steroid hormone receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells.

EXAMPLE 4 Preparation of Medicant-Estradiol 109

Analog 106 (FIG. 20EB) (54.5 mg, 0.15 mmol, 1 equiv.), β-estradiol (40.5mg, 0.15 mmol), and DMAP (1.8 mg, 0.015 mmol, 0.1 equiv.) were dissolvedin CH₂Cl₂ (5 mL) at 0° C. To this solution was added CH₂Cl₂ solution ofDCC (165 μL, 1 M, 0.165 mmol, 1.1 equiv.). The mixture was raised toroom temperature after 0.5 h. After another 2 h, the mixture wasfiltered. The filtrate was washed with dilute HCl (1.5%), saturatedNaHCO₃ and brine in sequence. The organic phase was dried andevaporated. The residue was eluted through a column (CH₂Cl₂/Methanol10:0.25) to give analog 109 (FIG. 20EE) (55 mg, 60%) as semisolid.

EXAMPLE 5 Preparation of Medicant-Estrone 110.

Analog 038 (FIG. 20BL) (68 mg, 0.2365 mmol), estrone (68.0 mg, 0.2160mmol) and DMAP (5 mg. 0.048 mmol) were dissolved in CH₂Cl₂ (8.0 mL) at0° C. To this solution was added CH₂Cl₂ solution of DCC (300 μL, 1 M,0.283 mmol), stirred for 30 minutes, allowed to warm to room temperaturethen stirred for 0.5 hours. The filtrate was washed with dilute HCl(1.5%), saturated NaHCO₃ and brine in sequence. The organic phase wasdried over Na₂SO₄, and evaporated. The residue was eluted through acolumn (100% CH₂Cl₂), fractions collected then eluted through a secondcolumn (CH₂Cl₂plus 0.5% methanol), to give analog 110 (FIG. 20EF) (40mg) as a solid.

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean±SD) for 110 (FIG. 20EF). MCF7 cells over expressestrogen alpha-receptors. MCF7 cells are preferentially killed by theacylfulvene-estrone analog 110 (FIG. 20EF) and to a lesser extent by theacylfulvene-estradiol analog 108 (FIG. 20ED) because estronepreferentially binds to alpha-receptor. In contrast, illudin M killedboth ER negative and ER positive cells to the same extent. The data inTable VII demonstrates that compounds 108 (FIG. 20ED) and 110 (FIG.20EF) are preferentially cytotoxic to cells expressing large numbers ofestrogen receptors on their surface.

EXAMPLE 6 Preparation of Medicant-Testosterone 111

Analog 038 (FIG. 20BL) (52.5 mg, 0.182 mmol), testosterone (50.0 mg,0.173 mmol) and DMAP (5 mg. 0.048 mmol) were dissolved in CH₂Cl₂ (8.0mL) at 0° C. To this solution was added CH₂Cl₂ solution of DCC (250 μL,1 M), stirred for 30 minutes, allowed to warm to room temperature thenstirred for 2 hours. The filtrate was washed with dilute HCl (1.5%),saturated NaHCO₃ and brine in sequence. The organic phase was dried overNa₂SO₄, and evaporated. The residue was eluted through a column (100%CH₂Cl₂ plus 0.5% methanol), to give analog 111 (FIG. 20EG) (15 mg) as asolid.

EXAMPLE 7 Preparation of Medicant-Androsterone 112

Analog 038 (FIG. 20BL) (29 mg), androsterone (25.0 mg) and DMAP (5 mg.0.048 mmol) were dissolved in CH₂Cl₂ (5.0 mL) at 0° C. To this solutionwas added CH₂Cl₂ solution of DCC (150 μL, 1 M), stirred for 30 minutes,allowed to warm to room temperature then stirred for 2 hours. Thefiltrate was washed with dilute HCl (1.5%), saturated NaHCO₃ and brinein sequence. The organic phase was dried over Na₂SO₄, and evaporated.The residue was eluted through a column (2:3 ethyl acetate: hexane) togive analog 112 (FIG. 20EH) (15 mg) as a solid.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a traditional linker 1240 to a steroid 1140 bind to receptorsfor the steroid and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, asteroid 1140 linked via a traditional linker 1240 to an illudin2 moiety1302 acts as an AM for the steroid hormone receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells factor receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a FSB linker 1220to an illudin2 moiety 1302 acts as an AM for the growth factor receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, agrowth factor 1120 linked via a FSB linker 1220 to an illudin2 moiety1302 acts as an AM for the growth factor receptor and directs theilludin2 moiety 1302 to tumors containing cells over-expressing thereceptor compared to non tumor cells.

FSB Linker to bind Illudin to a Steroid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to asteroid 1140 binds to receptors for the steroid and direct the illudin2moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the steroidhormone receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the steroid hormone receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, asteroid 1140 linked via a FSB linker 1220 to an illudin2 moiety 1302acts as an AM for the steroid hormone receptor and directs the illudin2moiety 1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as an AM for the steroidhormone receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the steroid hormone receptor anddirects the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

FSB linker to bind Illudin to an Anti-angiogenic peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaa FSB linker 1220 to an anti-angiogenic peptide 1130 binds to receptorsfor the anti-angiogenic peptide and directs the illudin2 moiety 1302 tocell populations expressing the receptor. In an embodiment of thepresent invention, an anti-angiogenic peptide 1130 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for theanti-angiogenic peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, an anti-angiogenic peptide 1130 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the peptidereceptor and directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via a FSB linker 1220 to an illudin2moiety 1302 acts as an AM for the anti-angiogenic peptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via a FSB linker 1220 to an illudin2moiety 1302 acts as an AM for the peptide receptor and directs theilludin2 moiety 1302 to tumors containing cells expressing the receptor.In an embodiment of the present invention, an anti-angiogenic peptide1130 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as anAM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

FSB Linker to bind Illudin to an Integrin binding peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaa FSB linker 1220 to an integrin binding peptide 1150 binds to receptorsfor the integrin binding peptide and directs the illudin2 moiety 1302 tocell populations expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked via a FSBlinker 1220 to an illudin2 moiety 1302 acts as an AM for the integrinbinding peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as an AM for the integrin bindingpeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding peptide 1150 linked FSB linker1220 to an illudin2 moiety 1302 acts as an AM for the integrin bindingpeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

FSB Linker to bind Illudin to a Pro-peptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a FSB linker 1220to a pro-peptide 1160 is cleaved by an enzyme 1165 to generate thepeptide 1161 and thereafter binds to receptors for the peptide anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a pro-peptide 1160linked via a FSB linker 1220 to an illudin2 moiety 1302 is cleaved by anenzyme 1165 and thereafter the peptide 1161 acts as a ligand for thepeptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a pro-peptide 1160 linked via a FSB linker 1220 to anilludin2 moiety 1302 is cleaved by an enzyme 1165 and thereafter thepeptide 1161 directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, apro-peptide 1160 linked via a FSB linker 1220 to an illudin2 moiety 1302is cleaved by an enzyme 1165 and thereafter the peptide 1161 acts as anAM for the peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, a pro-peptide 1160 linked via a FSB linker 1220to an illudin2 moiety 1302 is cleaved by an enzyme 1165 and thereafterthe peptide 1161 directs the illudin2 moiety 1302 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, a pro-peptide 1160 linked via a FSB linker 1220 to anilludin2 moiety 1302 is cleaved by an enzyme 1165 and thereafter thepeptide 1161 directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

FSB Linker to bind Illudin to a Glycopeptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a FSB linker 1220to a glycopeptide 1170 with biological activity binds to receptors forthe glycopeptide 1170 and directs the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as aligand for the glycopeptide receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, a glycopeptide 1170 linked via aFSB linker 1220 to an illudin2 moiety 1302 acts as an AM for theglycopeptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via a FSB linker 1220 toan illudin2 moiety 1302 acts as an AM for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as anAM for the glycopeptide receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

FSB Linker to bind Illudin to a Lipid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to alipid 1180 with biological activity binds to receptors for the lipid1180 and directs the illudin2 moiety 1302 to cell populations expressingthe lipid. In an embodiment of the present invention, a lipid 1180linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as a ligandfor the lipid receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a lipid 1180 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the lipid receptor and directsthe illudin2 moiety 1302 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a lipid 1180 linkedvia a FSB linker 1220 to an illudin2 moiety 1302 acts as an AM for thelipid receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a lipid 1180 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the lipid receptor and directsthe illudin2 moiety 1302 to tumors containing cells over-expressing thereceptor compared to non tumor cells.

FSB Linker to bind Illudin to a Peptide. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a FSB linker 1220 to apeptide 1190 with biological activity binds to the peptide receptor anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as a ligandfor the peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as a ligand for the peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as an AMfor the peptide receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via a FSB linker 1220 to anilludin2 moiety 1302 acts as an AM for the peptide receptor and directsthe illudin2 moiety 1302 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a FSB linker 1220 to an illudin2 moiety 1302 acts as an AMfor the peptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Linker to bind Illudin2 to a Glycopeptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a traditionallinker 1240 to a glycopeptide 1170 with biological activity binds toreceptors for the glycopeptide 1170 and directs the illudin2 moiety 1302to cell populations expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the glycopeptidereceptor and directs the illudin2 moiety 1302 to tissues containingcells expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via a traditional linker 1240 toan illudin2 moiety 1302 acts as a ligand for the glycopeptide receptorand directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor.

Linker to bind Illudin2 to a Lipid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a lipid 1180 with biological activity binds to receptors for thelipid 1180 and directs the illudin2 moiety 1302 to cell populationsexpressing the lipid receptor or lipid binding protein. In an embodimentof the present invention, a lipid 1180 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the lipid receptorand directs the illudin2 moiety 1302 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, alipid 1180 linked via a traditional linker 1240 to an illudin2 moiety1302 acts as a ligand for the lipid receptor and directs the illudin2moiety 1302 to tumors containing cells expressing the receptor. Linkerto bind Illudin2 to a Growth Factor In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a growth factor 1120 with biological activity binds to receptors forthe growth factor 1120 and directs the illudin2 moiety 1302 to cellpopulations expressing the growth factor receptor. In an embodiment ofthe present invention, a growth factor 1120 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for the growthfactor receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a growth factor 1120 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the growth factorreceptor and directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor.

Linker to bind Illudin2 to an anti-angiogenic peptide. In an embodimentof the present invention, an illudin2 moiety 1302 linked via atraditional linker 1240 to an anti-angiogenic peptide 1130 withbiological activity binds to receptors for the anti-angiogenic peptide1130 and directs the illudin2 moiety 1302 to cell populations expressingthe anti-angiogenic peptide receptor. In an embodiment of the presentinvention, an anti-angiogenic peptide 1130 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for theanti-angiogenic peptide receptor and directs the illudin2 moiety 1302 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, an anti-angiogenic peptide 1130 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe anti-angiogenic peptide receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to a Steroid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a steroid 1140 with biological activity binds to receptors for thesteroid 1140 and directs the illudin2 moiety 1302 to cell populationsexpressing the steroid receptor. In an embodiment of the presentinvention, a steroid 1140 linked via a traditional linker 1240 to anilludin2 moiety 1302 acts as a ligand for the steroid receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a steroid 1140linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asa ligand for the steroid receptor and directs the illudin2 moiety 1302to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to an Integrin binding protein. In an embodimentof the present invention, an illudin2 moiety 1302 linked via atraditional linker 1240 to an integrin binding protein 1150 withbiological activity binds to receptors for the integrin binding protein1150 and directs the illudin2 moiety 1302 to cell populations expressingthe integrin binding protein receptor. In an embodiment of the presentinvention, an integrin binding protein 1150 linked via a traditionallinker 1240 to an illudin2 moiety 1302 acts as a ligand for the integrinbinding protein receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an integrin binding protein 1150 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe integrin binding protein receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor.

Linker to bind Illudin2 to Folate. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to folate 1185 binds to receptors for the folate 1185 and directs theilludin2 moiety 1302 to cell populations expressing the folate receptor.In an embodiment of the present invention, folate 1185 linked via atraditional linker 1240 to an illudin2 moiety 1302 acts as a ligand forthe folate receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, folate 1185 linked via a traditional linker 1240 toan illudin2 moiety 1302 acts as a ligand for the folate receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor.

Linker to bind Illudin2 to a Peptide. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a traditional linker 1240to a peptide 1190 with biological activity binds to the peptide receptorand directs the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a traditional linker 1240 to an illudin2 moiety 1302 acts asa ligand for the peptide receptor and directs the illudin2 moiety 1302to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, a peptide 1190 linked via a traditional linker1240 to an illudin2 moiety 1302 acts as a ligand for the peptidereceptor and directs the illudin2 moiety 1302 to tumors containing cellsexpressing the receptor.

Mal1 linker 1211. Synthesis of mono-protected linkers. Maleimide orMaleic derivatives of acylfulvenes, Illudins and Syn-illudins can reactdirectly with thiol groups on antibodies, proteins or with primaryamines. When the Mal1 linker is used there is no need to attach theacylfulvene or illudin analog to linker as it is already incorporatedinto the analog (see FIG. 2S, FIG. 2T, FIG. 2U, and FIG. 2V). BothIlludin and Acylfulvene derivatives have been synthesized (e.g., analog189 (FIG. 20HF), analog 190 (FIG. 20HG), analog 217 (FIG. 20IH), andanalog 218 (FIG. 20II), see also FIG. 19), and data demonstrating theirin vitro activity and selectivity towards cells expressing a specificsurface antigen are shown in FIG. 19.

EXAMPLE 12

In an embodiment of the present invention, AMC's 189 (FIG. 20HF), 190(FIG. 20HG), 217 (FIG. 20IH), 218 (FIG. 20II) incorporating Mal1 linkerswere synthesized. Unexpectedly, the AMC 189 (FIG. 20HF), 190 (FIG.20HG), 217 (FIG. 20IH), 218 (FIG. 20II) were found to be cytotoxic withnM activity (see Table XV).

In an embodiment of the present invention, an AMC in which analog 218(FIG. 20IH) bound to an antibody using the Mal1 linker (FIG. 19) showssuperior activity compared to current antibodies medicinally used (e.g.Herceptin). Table XV shows the cytotoxicity data for AMC's 189 (FIG.20HF), 190 (FIG. 20HG), 217 (FIG. 20IH), 218 (FIG. 20II) incorporatingthe Mal1 linker.

In an embodiment of the present invention, the Mal1 linker was attachedto the acylfulvene. In this manner, the medicant-linker will binddirectly to sulfhydryl groups on an AM, e.g., antibody or peptidescontaining a cysteine (with a sulfhydryl group). This novelmedicant-linker allows the generation of toxin-peptide conjugates thatcan be cleaved by enzymes. Alternatively toxin-peptide conjugates can beprepared that will bind directly to tumor associated antigens (PMSA),specific integrins, or anti-angiogenic peptides.

Mal linker to bind Illudin to an Antibody. In an embodiment of thepresent invention, an illudin1 moiety 1301 linked via a Mal1 linker 1210to an antibody 1110 binds to a receptor to which the antibody 1110 wasprepared and directs the illudin1 moiety 1301 to cell populationsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 linked via Mal1 linker 1210 to an illudin1 moiety 1301acts as an AM for a receptor and directs the illudin1 moiety 1301 totissues containing cells expressing the receptor. In an embodiment ofthe present invention, an antibody 1110 linked via Mal1 linker 1210 toan illudin1 moiety 1301 acts as an AM for a receptor and directs theilludin1 moiety 1301 to tumors containing cells expressing the receptor.In an embodiment of the present invention, an antibody 1110 linked viaMal1 linker 1210 to an illudin1 moiety 1301 acts as an AM for a receptorand directs the illudin1 moiety 1301 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

In an embodiment of the present invention, an illudin2 moiety 1302linked via a Mal1 linker 1211 to an antibody 1110 binds to a receptor towhich the antibody 1110 was prepared and directs the illudin2 moiety1302 to cell populations expressing the receptor. In an embodiment ofthe present invention, an antibody 1110 linked via Mal1 linker 1211 toan illudin2 moiety 1302 acts as an AM for a receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, an antibody 1110linked via Mal1 linker 1211 to an illudin2 moiety 1302 acts as an AM fora receptor and directs the illudin2 moiety 1302 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, an antibody 1110 linked via Mal1 linker 1211 to an illudin1moiety 1301 acts as an AM for a receptor and directs the illudin1 moiety1301 to tumors containing cells over-expressing the receptor compared tonon tumor cells.

Mal1 linker to bind a Medicant to an Antibody. In an embodiment of thepresent invention, an antibody 1110 is bound to a medicant 1350 with aMal1 linker 1210. In an embodiment of the present invention, an antibody1110 directs the medicant 1350 to cell populations expressing thereceptor. In an embodiment of the present invention, the antibody 1110with a Mal1 linker 1210 to the medicant 1350 acts as a ligand for areceptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with a Mal1 linker 1210 to the medicant 1350 acts as aligand for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor.

Mal1 linker to bind Illudin to Growth factor. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a Mal1 linker 1210to a growth factor 1120 binds to the growth factor receptor and directthe illudin2 moiety 1302 to cell populations expressing the receptor. Inan embodiment of the present invention, a growth factor 1120 linked viaa Mal1 linker 1210 to an illudin2 moiety 1302 acts as an AM for thegrowth factor receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a growth factor 1120 linked via a Mal1 linker 1210 toan illudin2 moiety 1302 act as an AM for the growth factor receptor anddirect the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a growth factor1120 linked via a Mal1 linker 1210 to an illudin2 moiety 1302 act as anAM for the growth factor receptor and direct the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells. In an embodiment of the present invention, an illudin1moiety 1301 linked via a Mal1 linker 1210 to a growth factor 1120 bindsto the growth factor receptor and direct the illudin1 moiety 1301 tocell populations expressing the receptor. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a Mal1 linker 1211to a growth factor 1120 binds to the growth factor receptor and directthe illudin2 moiety 1302 to cell populations expressing the receptor.

Mal1 linker to bind Illudin2 to a Steroid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a Mal1 linker 1210to a steroid 1140 bind to receptors for the steroid and direct theilludin2 moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via a Mal1linker 1210 to an illudin2 moiety 1302 acts as an AM for the steroidhormone receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via a Mal1 linker 1210 to anilludin2 moiety 1302 acts as an AM for the steroid hormone receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a steroid 1140linked via a Mal1 linker 1210 to an illudin2 moiety 1302 acts as an AMfor the steroid hormone receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

Mal1 linker to bind Illudin to Anti-Angiogenic Peptide. In an embodimentof the present invention, an illudin2 moiety 1302 linked via a Mal1linker 1210 to an anti-angiogenic peptide 1130 bind to receptors for theanti-angiogenic peptide and direct the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, an anti-angiogenic peptide 1130 linked via Mal1 linker 1210to an illudin2 moiety 1302 acts as an AM for the peptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via Mal1 linker 1210 to an illudin2moiety 1302 acts as an AM for the anti-angiogenic peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, ananti-angiogenic peptide 1130 linked via Mal1 linker 1210 to an illudin2moiety 1302 acts as an AM for the anti-angiogenic peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Mal1 linker to bind Illudin to Integrin Binding Peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaa Mal1 linker 1210 to an integrin binding peptide 1150 bind to receptorsfor the peptide and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, anintegrin binding peptide 1150 linked via Mal1 linker 1210 to an illudin2moiety 1302 acts as an AM for the peptide receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, an integrin bindingpeptide 1150 linked via Mal1 linker 1210 to an illudin2 moiety 1302 actsas an AM for the peptide receptor and directs the illudin2 moiety 1302to tumors containing cells expressing the receptor. In an embodiment ofthe present invention, an integrin binding peptide 1150 linked via Mal1linker 1210 to an illudin2 moiety 1302 acts as an AM for the peptidereceptor and directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Mal1 linker to bind Illudin to Pro-Peptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a Mal1 linker 1210to a pro-peptide cleavable by a protease 1165 binds to receptors for thepeptide and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, apeptide cleaved by a protease 1165 linked via Mal1 linker 1210 to anilludin2 moiety 1302 acts as an AM for the peptide receptor and directsthe illudin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a peptide cleavedby a protease 1165 linked via Mal1 linker 1210 to an illudin2 moiety1302 acts as an AM for the peptide receptor and directs the illudin2moiety 1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, a peptide cleaved by a protease1165 linked via Mal1 linker 1210 to an illudin2 moiety 1302 acts as anAM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

Mal1 linker to bind Illudin to a Glycopeptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via a Mal1 linker 1210to a glycopeptide 1170 with biological activity binds to receptors forthe glycopeptide and directs the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via a Mal1 linker 1211 to anilludin2 moiety 1302 acts as a ligand for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tissues containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via a Mal1 linker 1211 to an illudin2 moiety 1302 acts as aligand for the glycopeptide receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, a glycopeptide 1170 linked via aMal1 linker 1211 to an illudin2 moiety 1302 acts as a ligand for theglycopeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Mal1 linker to bind Illudin to a Lipid. In an embodiment of the presentinvention, an illudin2 moiety 1302 linked via a Mal1 linker 1210 to alipid 1180 with biological activity binds to receptors for the lipid1180 and directs the illudin2 moiety 1302 to cell populations expressingthe lipid. In an embodiment of the present invention, a lipid 1180linked via a Mal1 linker 1210 to an illudin2 moiety 1302 acts as an AMfor the lipid receptor and directs the illudin2 moiety 1302 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a lipid 1180 linked via a Mal1 linker 1210 to anilludin2 moiety 1302 acts as an AM for the lipid receptor and directsthe illudin2 moiety 1302 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a lipid 1180 linkedvia a Mal1 linker 1210 to an illudin2 moiety 1302 acts as an AM for thelipid receptor and directs the illudin2 moiety 1302 to tumors containingcells over-expressing the receptor compared to non tumor cells.

Mal1 linker to bind Illudin to a Peptide. In an embodiment of thepresent invention, an illudin1 moiety 1301 linked via a Mal1 linker 1210to a peptide 1190 with biological activity binds to the peptide receptorand directs the illudin1 moiety 1301 to cell populations expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a Mal1 linker 1210 to an illudin1 moiety 1301 acts as an AMfor the peptide receptor and directs the illudin1 moiety 1301 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via a Mal1 linker 1210 to anilludin1 moiety 1301 acts as an AM for the peptide receptor and directsthe illudin1 moiety 1301 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via a Mal1 linker 1210 to an illudin1 moiety 1301 acts as an AMfor the peptide receptor and directs the illudin1 moiety 1301 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Mal1 linker to bind an Antibody to Medicant. In an embodiment of thepresent invention, an antibody 1110 is bound to a medicant 1350 with aMal1 linker 1210. In an embodiment of the present invention, an antibody1110 directs the medicant 1350 to cell populations expressing thereceptor. In an embodiment of the present invention, the antibody 1110with a Mal1 linker 1210 to the medicant 1350 acts as an AM for areceptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with a Mal1 linker 1210 to the medicant 1350 acts as an AMfor a receptor and directs the medicant 1350 to tumors containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with a Mal1 linker 1210 to the medicant 1350 acts as an AMfor a receptor and directs the medicant 1350 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Mal1 linker to bind an Antibody to Protein Toxins. In an embodiment ofthe present invention, an antibody 1110 is bound to a protein toxin 1330with a Mal1 linker 1210. In an embodiment of the present invention, anantibody 1110 directs the protein toxin 1330 to cell populationsexpressing the receptor. In an embodiment of the present invention, theantibody 1110 with a Mal1 linker 1210 to a protein toxin 1330 acts as anAM for a receptor and directs the protein toxin 1330 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, an antibody 1110 with a Mal1 linker 1210 to theprotein toxin 1330 acts as an AM for a receptor and directs the proteintoxin 1330 to tumors containing cells expressing the receptor. In anembodiment of the present invention, an antibody 1110 with a Mal1 linker1210 to the protein toxin 1330 acts as an AM for a receptor and directsthe protein toxin 1330 to tumors containing cells over-expressing thereceptor compared to non tumor cells.

Mal1 linker to bind Growth factor to a Protein Toxin. In an embodimentof the present invention, a growth factor 1120 is bound to a proteintoxin 1330 with a Mal1 linker 1210. In an embodiment of the presentinvention, the growth factor 1120 directs the protein toxin 1330 to cellpopulations expressing the receptor to the growth factor 1120. In anembodiment of the present invention, the growth factor 1120 with a Mal1linker 1210 to a protein toxin 1330 acts as an AM for a receptor to thegrowth factor 1120 and directs the protein toxin 1330 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, the growth factor 1120 with a Mal1 linker 1210 to theprotein toxin 1330 acts as an AM for the growth factor receptor anddirects the protein toxin 1330 to tumors containing cells expressing thereceptor for the growth factor 1120. In an embodiment of the presentinvention, the growth factor 1120 with a Mal1 linker 1210 to the proteintoxin 1330 acts as an AM for the growth factor receptor and directs theprotein toxin 1330 to tumors containing cells expressing the receptorfor the growth factor 1120.

Mal1 linker to bind Growth factor to a Medicant. In an embodiment of thepresent invention, a growth factor 1120 is bound to a medicant 1350 witha Mal1 linker 1210. In an embodiment of the present invention, thegrowth factor 1120 directs the medicant 1350 to cell populationsexpressing the receptor to the growth factor 1120. In an embodiment ofthe present invention, the growth factor 1120 with a Mal1 linker 1210 toa medicant 1350 acts as an AM for a receptor to the growth factor 1120and directs the medicant 1350 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, the growth factor1120 with a Mal1 linker 1210 to the medicant 1350 acts as an AM for areceptor and directs the medicant 1350 to tumors containing cellsexpressing the receptor for the growth factor 1120. In an embodiment ofthe present invention, the growth factor 1120 with a Mal1 linker 1210 tothe medicant 1350 acts as an AM for a receptor and directs the medicant1350 to tumors containing cells expressing the receptor for the growthfactor 1120.

Synthesis of Linkers. The synthesis of medicant moieties bound tolinkers can be carried out using the following strategies: React R—NH₂with H—N═C═S to form isothiourea R—NH—C(═S)—NH₂. React R—NH₂ withH—N═C═O to form isourea R—NH—C(═O)—NH₂. React R—NH₂ with acyl azide toform RC(═O)NHR. React R—NH₂ with NHS ester to form RC(═O)NHR. Reactamine with sulfonyl chloride to form sulfonamide bond R(S(═O)(═O)NHR.React amine with imidoester to form amidine linkage RCH₂C(═NH₂)NHR.React amine with succinic acid to make amide bond with carboxylate ion.React imidoester with amine to form amidine bond RCH₂C(═NH₂+)NHR (seealso Table X).

FSB Linker to bind a Medicant to a Steroid. In an embodiment of thepresent invention, a steroid 1140 is bound to a medicant 1350 with a FSBlinker 1220. In an embodiment of the present invention, a steroid 1140directs the medicant 1350 to cell populations expressing the receptor.In an embodiment of the present invention, the steroid 1140 bound with aFSB linker 1220 to the medicant 1350 acts as an AM for a receptor anddirects the medicant 1350 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a steroid 1140bound with a FSB linker 1220 to the medicant 1350 acts as an AM for asteroid receptor and directs the medicant 1350 to tumors containingcells expressing the steroid receptor. In an embodiment of the presentinvention, a steroid 1140 bound with a FSB linker 1220 to the medicant1350 acts as an AM for a steroid receptor and directs the medicant 1350to tumors containing cells over-expressing the steroid receptor comparedto non tumor cells.

FSB Linker to bind a Medicant to an anti-angiogenic peptide. In anembodiment of the present invention, an anti angiogenic peptide 1130 isbound to a medicant 1350 with a FSB linker 1220. In an embodiment of thepresent invention, the anti angiogenic peptide 1130 directs the medicant1350 to cell populations expressing the receptor to the anti angiogenicpeptide 1130. In an embodiment of the present invention, the antiangiogenic peptide 1130 bound with a FSB linker 1220 to a medicant 1350acts as an AM for a receptor to the anti angiogenic peptide 1130 anddirects the medicant 1350 to tissues containing cells expressing theanti angiogenic peptide receptor. In an embodiment of the presentinvention, the anti angiogenic peptide 1130 bound with a FSB linker 1220to the medicant 1350 acts as an AM for a receptor to the anti angiogenicpeptide and directs the medicant 1350 to tumors containing cellsexpressing the receptor for the anti angiogenic peptide 1130. In analternative embodiment of the present invention, the anti angiogenicpeptide 1130 bound with a FSB linker 1220 to the medicant 1350 acts asan AM for a receptor and directs the medicant 1350 to tumors containingcells over expressing the receptor for the anti angiogenic peptide 1130compared to non-tumor cells. In an alternative embodiment of the presentinvention, the anti angiogenic peptide 1130 bound with a FSB linker 1220to the medicant 1350 acts as an AM for a receptor and directs themedicant 1350 to tumors containing cells over expressing the receptorfor the anti angiogenic peptide 1130 compared to non-tumor cells.

FSB Linker to bind a Medicant to an Integrin Binding Peptide. In anembodiment of the present invention, an integrin binding peptide 1150 isbound to a medicant 1350 with a FSB linker 1220. In an embodiment of thepresent invention, an integrin binding peptide 1150 directs the medicant1350 to cell populations expressing integrin. In an embodiment of thepresent invention, the integrin binding peptide 1150 bound with a FSBlinker 1220 to the medicant 1350 acts as an AM for integrin and directsthe medicant 1350 to tissues containing cells expressing integrin. In anembodiment of the present invention, an integrin binding peptide 1150bound with a FSB linker 1220 to the medicant 1350 acts as an AM forintegrin and directs the medicant 1350 to tumors containing cellsexpressing an integrin. In an embodiment of the present invention, anintegrin binding peptide 1150 bound with a FSB linker 1220 to themedicant 1350 acts as an AM for an integrin and directs the medicant1350 to tumors containing cells over-expressing the integrin compared tonon tumor cells.

FSB Linker to bind a Medicant to a pro-peptide. In an embodiment of thepresent invention, an pro-peptide 1160 is bound to a medicant 1350 witha FSB linker 1220. In an embodiment of the present invention, thepro-peptide 1160 directs the medicant 1350 to cell populationsexpressing the receptor to the peptide. In an embodiment of the presentinvention, the pro-peptide 1160 bound with a FSB linker 1220 to amedicant 1350 acts is cleaved by an enzyme 1165 to generate the peptide1161 which acts as an AM for the receptor to the peptide 1161 anddirects the medicant 1350 to tissues containing cells expressing thepeptide receptor. In an embodiment of the present invention, thepro-peptide 1160 bound with a FSB linker 1220 to the medicant 1350 actsas an AM for a receptor to the peptide 1161 and directs the medicant1350 to tumors containing cells expressing the peptide receptor. In analternative embodiment of the present invention, the pro-peptide 1160bound with a FSB linker 1220 to the medicant 1350 acts as an AM for areceptor and directs the medicant 1350 to tumors containing cells overexpressing the receptor for the peptide 1161 compared to non-tumorcells. In an alternative embodiment of the present invention, thepro-peptide 1160 bound with a FSB linker 1220 to the medicant 1350 iscleaved by an enzyme 1165 to generate the peptide 1161 which acts as anAM for a receptor and directs the medicant 1350 to tumors containingcells over expressing the receptor for the peptide 1161 compared tonon-tumor cells.

FSB Linker to bind a Medicant to a Peptide. In an embodiment of thepresent invention, a peptide 1190 is bound to a medicant 1350 with a FSBlinker 1220. In an embodiment of the present invention, a peptide 1190directs the medicant 1350 to cell populations expressing receptor forthe peptide. In an embodiment of the present invention, the peptide 1190bound with a FSB linker 1220 to the medicant 1350 acts as an AM for areceptor for the peptide and directs the medicant 1350 to tissuescontaining cells expressing the receptor for the peptide. In anembodiment of the present invention, a peptide 1190 bound with a FSBlinker 1220 to the medicant 1350 acts as an AM for a receptor for thepeptide and directs the medicant 1350 to tumors containing cellsexpressing the receptor for the peptide. In an embodiment of the presentinvention, a peptide 1190 bound with a FSB linker 1220 to the medicant1350 acts as an AM for a receptor for the peptide and directs themedicant 1350 to tumors containing cells over-expressing the receptorfor the peptide compared to non tumor cells.

FSB Linker to bind a Medicant to a Glycopeptide. In an embodiment of thepresent invention, a glycopeptide 1170 is bound to a medicant 1350 witha FSB linker 1220. In an embodiment of the present invention, aglycopeptide 1170 directs the medicant 1350 to cell populationsexpressing a receptor for the glycopeptide. In an embodiment of thepresent invention, the glycopeptide 1170 bound with a FSB linker 1220 tothe medicant 1350 acts as an AM for a glycopeptide receptor and directsthe medicant 1350 to tissues containing cells expressing theglycopeptide receptor. In an embodiment of the present invention, aglycopeptide 1170 bound with a FSB linker 1220 to the medicant 1350 actsas an AM for a glycopeptide receptor and directs the medicant 1350 totumors containing cells expressing the glycopeptide receptor. In anembodiment of the present invention, a glycopeptide 1170 bound with aFSB linker 1220 to the medicant 1350 acts as an AM for a glycopeptidereceptor and directs the medicant 1350 to tumors containing cellsover-expressing the glycopeptide receptor compared to non tumor cells.

FSB Linker to bind a Medicant to a Lipid. In an embodiment of thepresent invention, a lipid 1180 is bound to a medicant 1350 with a FSBlinker 1220. In an embodiment of the present invention, the lipid 1180directs the medicant 1350 to cell populations expressing the receptor tothe lipid. In an embodiment of the present invention, the lipid 1180bound with a FSB linker 1220 to a medicant 1350 directs the medicant1350 to tissues containing cells expressing a receptor activated by thelipid 1180. In an embodiment of the present invention, the lipid 1180bound with a FSB linker 1220 to the medicant 1350 directs the medicant1350 to tumors containing cells expressing the lipid receptor. In analternative embodiment of the present invention, the lipid 1180 boundwith a FSB linker 1220 to the medicant 1350 acts as an AM for a receptorand directs the medicant 1350 to tumors containing cells over expressingthe receptor activated by the lipid 1180 compared to non-tumor cells. Inan alternative embodiment of the present invention, the lipid 1180 boundwith a FSB linker 1220 to the medicant 1350 acts as an AM for a receptorand directs the medicant 1350 to tumors containing cells over expressingthe receptor activated by the lipid 1180 compared to non-tumor cells.

FSB Linker to bind a Medicant to Folate. In an embodiment of the presentinvention, folate 1185 is bound to a medicant 1350 with a FSB linker1220. In an embodiment of the present invention, the folate 1185 directsthe medicant 1350 to cell populations expressing the folate receptor. Inan embodiment of the present invention, the folate 1185 bound with a FSBlinker 1220 to a medicant 1350 directs the medicant 1350 to tissuescontaining cells expressing the folate receptor. In an embodiment of thepresent invention, the folate 1185 bound with a FSB linker 1220 to themedicant 1350 directs the medicant 1350 to tumors containing cellsexpressing the folate receptor. In an alternative embodiment of thepresent invention, the folate 1185 bound with a FSB linker 1220 to themedicant 1350 acts as an AM for the folate receptor and directs themedicant 1350 to tumors containing cells over expressing the folatereceptor compared to non-tumor cells. In an alternative embodiment ofthe present invention, the folate 1185 bound with a FSB linker 1220 tothe medicant 1350 acts as an AM for the folate receptor and directs themedicant 1350 to tumors containing cells over expressing the folatereceptor compared to non-tumor cells.

FSB linker to bind a Medicant to an Antibody. In an embodiment of thepresent invention, an antibody 1110 is bound to a medicant 1350 with aFSB linker 1220. In an embodiment of the present invention, an antibody1110 directs the medicant 1350 to cell populations expressing thereceptor. In an embodiment of the present invention, the antibody 1110with a FSB linker 1220 to the medicant 1350 acts as a ligand for areceptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with a FSB linker 1220 to the medicant 1350 acts as aligand for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor.

FSB linker to bind a Medicant to a Growth factor. In an embodiment ofthe present invention, a growth factor 1120 is bound to a medicant 1350with a FSB linker 1220. In an embodiment of the present invention, thegrowth factor 1120 directs the medicant 1350 to cell populationsexpressing the receptor to the growth factor 1120. In an embodiment ofthe present invention, the growth factor 1120 with a FSB linker 1220 toa medicant 1350 acts as a ligand for a receptor to the growth factor1120 and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, thegrowth factor 1120 with a FSB linker 1220 to the medicant 1350 acts as aligand for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor for the growth factor 1120.

FSB linker to bind a Medicant to an Affinity Moiety. In an embodiment ofthe present invention, an AM 1100 is bound to a medicant 1350 with a FSBlinker 1220. In an embodiment of the present invention, the AM 1100directs the medicant 1350 to cell populations expressing a receptor orbinding protein for the AM. In an embodiment of the present invention,the AM 1100 bound with a FSB linker 1220 to a medicant 1350 directs themedicant 1350 to tissues containing cells expressing the receptor orbinding protein. In an embodiment of the present invention, the AM 1100bound with a FSB linker 1220 to the medicant 1350 directs the medicant1350 to tumors containing cells expressing the receptor or bindingprotein. In an alternative embodiment of the present invention, the AM1100 bound with a FSB linker 1220 to the medicant 1350 acts as an AM forthe receptor or binding protein and directs the medicant 1350 to tumorscontaining cells over expressing the receptor or binding proteincompared to non-tumor cells. In an alternative embodiment of the presentinvention, the AM 1100 bound with a FSB linker 1220 to the medicant 1350acts as an AM for the receptor and/or binding protein and directs themedicant 1350 to tumors containing cells over expressing the receptorand/or binding protein compared to non-tumor cells.

FSB Linker to bind Illudin2 to Folate. In an embodiment of the presentinvention, folate 1185 is bound to an illudin2 moiety 1302 with a FSBlinker 1220. In an embodiment of the present invention, the illudin2moiety 1302 linked via a FSB linker 1220 to folate 1185 binds toreceptors for the folate 1185 and directs the illudin2 moiety 1302 tocell populations expressing the folate receptor. In an embodiment of thepresent invention, the folate 1185 directs the illudin2 moiety 1302 tocell populations expressing the folate receptor. In an embodiment of thepresent invention, the folate 1185 bound with a FSB linker 1220 to anilludin2 moiety 1302 directs the illudin2 moiety 1302 to tissuescontaining cells expressing the folate receptor. In an embodiment of thepresent invention, the folate 1185 bound with a FSB linker 1220 to theilludin2 moiety 1302 directs the an illudin2 moiety 1302 to tumorscontaining cells expressing the folate receptor. In an alternativeembodiment of the present invention, the folate 1185 bound with a FSBlinker 1220 to the illudin2 moiety 1302 acts as an AM for the folatereceptor and directs the an illudin2 moiety 1302 to tumors containingcells over expressing the folate receptor compared to non-tumor cells.In an alternative embodiment of the present invention, the folate 1185bound with a FSB linker 1220 to the illudin2 moiety 1302 acts as an AMfor the folate receptor and directs an illudin2 moiety 1302 to tumorscontaining cells over expressing the folate receptor compared tonon-tumor cells.

Azlactone linker. Reactions of carbodiimides such asdicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) with acarboxylic acid yields a highly reactive O-acylisourea. Duringartificial protein synthesis (such as Fmoc solid-state synthesizers),the C-terminus is often used as the attachment site on which the aminoacid monomers are added. To enhance the electrophilicity of carboxylategroup, the negatively charged oxygen must first be “activated” into abetter leaving group and carbodiimides can be used for this purpose. Thenegatively charged oxygen will act as a nucleophile, attacking thecentral carbon in DCC. DCC is temporarily attached to the formercarboxylate group (which is now an ester group), making nucleophilicattack by an amino group (on the attaching amino acid) to the formerC-terminus (carbonyl group) more efficient.

When the Illudin, Syn-Illudin, or Acylfulvene carboxylic acid analog isactivated by DCC or DIC in the presence of an amino acid theDCC-activated carboxylate will react with the amino acid to form anazlactone (FIG. 2P, FIG. 2Q, FIG. 2R, and FIG. 10). This aminoacid-derived azlactone will react with primary amines, undergo ringopening, and forms an amide bond.

EXAMPLE 13 Synthesis of Medicant 106

Illudin M (450 mg, 1.845 mmol, 1 equiv.), glutaric anhydride (2.10 g,18.45 mmol, 10 equiv.) and DMAP (171 mg, 1.4 mmol, 0.76 equiv.) weredissolved in CH₂Cl₂ (5 mL) at room temperature. After 3.5 hours themixture was taken up by CH₂Cl₂, which was washed with water, and brinein sequence. It was then dried and evaporated. The residue was elutedthrough a column (Hexane/EtOAc 4:1) to give analog 106 (see FIG. 20EB)(365 mg, 55%) as a liquid. UV (CHCl₃) λ, nm (ε): 309 (3387).

Analog 106 (FIG. 20EB) was generated from illudin M as outlined inExample 13. The carboxylic acid derivative was activated using DCC/DMAPto synthesize steroid AFC's 107 (FIG. 20EC) and 109 (FIG. 20EE). Inaddition, Irofulven carboxylic acid derivative, analog 038 (FIG. 20BL)was activated using DCC/DMAP to produce analogs 108 (FIG. 20ED), 110(FIG. 20EF), 111 (FIG. 20EG), and 112 (FIG. 20EH). In general,carboxylate group containing compounds can be activated using acarbodiimide in the presence of an amino acid to form an azlactone. Theazlactone formed will react spontaneously with primary amine groups onan amino acid, a peptide, an antibody, a protein, or another drug, andundergo ring opening with the formation of an amide bond. For proteins,antibodies and peptides the amino acids capable of reacting with theazlactone derivative includes arginine and lysine.

To form an Illudin derived azlactone active drug-linker moiety, eitheranalog 106 (FIG. 20EB) or analog 038 (FIG. 20BL) can be activated byDCC/DMAP in the presence of a small amino acid such as glycine to formthe azlactone. DCC cannot be added without the presence of an aminecontaining target (such as the glycine) or the activated carboxylatereacts with another carboxylate to form a symmetrical anhydride. Theazlactone formed will react spontaneously with primary amine groups on apeptide, an antibody, a protein, or a medicant.

EXAMPLE 14 Activation of Analog 038 (FIG. 20BL) by DCC to FormMedicant-azlactone

Part A: Production of Azlactone from carboxylate Acylfulvene analog:Analog 038 (FIG. 20BL) (58.5 mg, 0.2035 mmol), and DMAP (5 mg. 0.048mmol) were dissolved in CH₂Cl₂ (5.6 mL) at 0° C. The desired amino acid(such as glycine) is added in an equimolar amount. Note that amino acidshaving substitutions on the C4 carbon (such as alpha-methyl glycine or2-dimethylglycine) are preferred over conventional amino acids assubstitution cannot occur at the C4 position after ring-opening and allnucleophilic coupling reactions must occur at the C5 position, resultingonly in the desired amide-bond formation with the amine-containingmolecule. To this solution was added CH₂Cl₂ solution of DCC (250 μL, 1M, 0.244 mmol), stirred for 30 minutes, allowed to warm to roomtemperature then stirred for 1.5 hours. The filtrate was washed withdilute HCl (1.5%), saturated NaHCO₃ and brine in sequence. The organicphase was dried over Na₂SO₄, and evaporated. The residue was elutedthrough a column (100% CH₂Cl₂plus 0.5% methanol), to give the desiredazlactone analog as a solid. Part B: Coupling of Azlactone to theprotein component (reacting with primary amines on amino acids such asthe one on lysine): The typical protein coupling reaction consists ofthe Azlactone suspended in buffer [25 mM sodium phosphate, 150 mM NaCl(pH 7.5)] and the desired amount of protein (20 μg to 5.0 mg) is added.The mixture is gently rocked for 60 minutes, then the reactionterminated by the addition of the blocking reagent, 1.0 ml of 1.0 Methanolamine in 25 mM sodium pyrophosphate (titrated to pH 9.0 with HCI)Sample rocked gently for 5 minutes then the residual ethanolamineremoved by dialysis or chromatography using pH 7.5 phosphate-NaClbuffer.

EXAMPLE 15 Reaction of the Medicant-azlactone Product with an Antibody

The azlactone derivative generated in Example 14 (note that other aminoacids can be used in place of glycine) is then reacted with the desiredpeptide or protein or other compound containing a primary amino group ata 1:1 ratio in buffer (25 mM sodium phosphate, 150 mM sodium chloride,pH 7.5) with gentle rocking at room temperature for 60 minutes. Thereaction is terminated by the addition of 1.0 mL of 25 mM ethanolamine(titrated to pH 9.00) with rocking for 5 minutes at room temperature).The drug-azlactone-ligand product can be purified by columnchromatography or dialysis to remove the ethanolamine by-product.

Azlactone linker to bind Antibody to a Protein Toxin. In an embodimentof the present invention, an antibody 1110 is bound to a protein toxin1330 with an azlactone linker 1230. In an embodiment of the presentinvention, an antibody 1110 directs the protein toxin 1330 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, the antibody 1110 with an azlactone linker 1230 to a proteintoxin 1330 acts as an AM for a receptor and directs the protein toxin1330 to tissues containing cells expressing the receptor. In anembodiment of the present invention, an antibody 1110 with an azlactonelinker 1230 to the protein toxin 1330 acts as an AM for a receptor anddirects the protein toxin 1330 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, an antibody 1110with an azlactone linker 1230 to the protein toxin 1330 acts as an AMfor a receptor and directs the protein toxin 1330 to tumors containingcells over-expressing the receptor compared to non tumor cells.

Azlactone linker to bind Antibody to a Medicant. In an embodiment of thepresent invention, an antibody 1110 is bound to a medicant 1350 with anazlactone linker 1230. In an embodiment of the present invention, anantibody 1110 directs the medicant 1350 to cell populations expressingthe receptor. In an embodiment of the present invention, the antibody1110 with an azlactone linker 1230 to the medicant 1350 acts as an AMfor a receptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with an azlactone linker 1230 to the medicant 1350 acts asan AM for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, an antibody 1110 with an azlactone linker 1230 to themedicant 1350 acts as an AM for a receptor and directs the medicant 1350to tumors containing cells over-expressing the receptor compared to nontumor cells.

Azlactone linker to bind Growth Factor to a Protein Toxin. In anembodiment of the present invention, a growth factor 1120 is bound to aprotein toxin 1330 with an azlactone linker 1230. In an embodiment ofthe present invention, the growth factor 1120 directs the protein toxin1330 to cell populations expressing the receptor to the growth factor1120. In an embodiment of the present invention, the growth factor 1120with an azlactone linker 1230 to a protein toxin 1330 acts as an AM fora receptor to the growth factor 1120 and directs the protein toxin 1330to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, the growth factor 1120 with an azlactone linker1230 to the protein toxin 1330 acts as an AM for the growth factorreceptor and directs the protein toxin 1330 to tumors containing cellsexpressing the receptor for the growth factor 1120. In an embodiment ofthe present invention, the growth factor 1120 with an azlactone linker1230 to the protein toxin 1330 acts as an AM for the growth factorreceptor and directs the protein toxin 1330 to tumors containing cellsexpressing the receptor for the growth factor 1120.

Azlactone linker to bind Growth Factor to a Medicant. In an embodimentof the present invention, a growth factor 1120 is bound to a medicant1350 with an azlactone linker 1230. In an embodiment of the presentinvention, the growth factor 1120 directs the medicant 1350 to cellpopulations expressing the receptor to the growth factor 1120. In anembodiment of the present invention, the growth factor 1120 with anazlactone linker 1230 to a medicant 1350 acts as an AM for a receptor tothe growth factor 1120 and directs the medicant 1350 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, the growth factor 1120 with an azlactone linker 1230to the medicant 1350 acts as an AM for a receptor and directs themedicant 1350 to tumors containing cells expressing the receptor for thegrowth factor 1120. In an alternative embodiment of the presentinvention, the growth factor 1120 with an azlactone linker 1230 to themedicant 1350 acts as an AM for a receptor and directs the medicant 1350to tumors containing cells over expressing the receptor for the growthfactor 1120 compared to non-tumor cells. In an alternative embodiment ofthe present invention, the growth factor 1120 with an azlactone linker1230 to the medicant 1350 acts as an AM for a receptor and directs themedicant 1350 to tumors containing cells over expressing the receptorfor the growth factor 1120 compared to non-tumor cells.

Azlactone linker to bind Illudin2 to an Antibody. In an embodiment ofthe present invention, an antibody 1110 with an azlactone linker 1230 toan illudin2 moiety 1302 (see FIG. 2R for the azlactone linker 1230 onthe illudin2 1302 moiety) binds to a receptor to which the antibody 1110was prepared and directs the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 bound with an azlactone linker 1230 to an illudin2 moiety1302 acts as an AM for a receptor and directs the illudin2 moiety 1302to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, an antibody 1110 with an azlactone linker 1230bound to an illudin2 moiety 1302 acts as an AM for a receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, an antibody1110 with an azlactone linker 1230 bound to an illudin2 moiety 1302 actsas an AM for a receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Azlactone linker to bind Illudin2 to an Growth Factor. In an embodimentof the present invention, an illudin2 moiety 1302 linked via anazlactone linker 1230 to a growth factor 1120 binds to the growth factorreceptor and direct the illudin2 moiety 1302 to cell populationsexpressing the receptor. In an embodiment of the present invention, agrowth factor 1120 linked via an azlactone linker 1230 to an illudin2moiety 1302 acts as an AM for the growth factor receptor and directs theilludin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a growth factor1120 linked via an azlactone linker 1230 to an illudin2 moiety 1302 actsas an AM for the growth factor receptor and directs the illudin2 moiety1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, a growth factor 1120 linked via anazlactone linker 1230 to an illudin2 moiety 1302 acts as an AM for thegrowth factor receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Azlactone linker to bind Illudin2 to an Steroid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a steroid 1140 binds to receptors for the steroid anddirect the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via anazlactone linker 1230 to an illudin2 moiety 1302 acts as an AM for thesteroid hormone receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via an azlactone linker 1230 toan illudin2 moiety 1302 acts as an AM for the steroid hormone receptorand directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind Illudin2 to an Anti-Angiogenic Peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaan azlactone linker 1230 to an anti-angiogenic peptide 1130 binds toreceptors for the anti-angiogenic peptide and directs the illudin2moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, an anti-angiogenic peptide 1130linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the anti-angiogenic peptide receptor and directs the illudin2moiety 1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, an anti-angiogenic peptide 1130linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells expressing the receptor. In an embodiment of thepresent invention, an anti-angiogenic peptide 1130 linked via anazlactone linker 1230 to an illudin2 moiety 1302 acts as an AM for thepeptide receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Azlactone linker to bind Illudin2 to an Integrin Binding Peptide. In anembodiment of the present invention, an illudin2 moiety 1302 linked viaan azlactone linker 1230 to an integrin binding peptide 1150 binds toreceptors for the integrin binding peptide and directs the illudin2moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, an integrin binding peptide 1150linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the integrin binding peptide receptor and directs the illudin2moiety 1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, an integrin binding peptide 1150linked azlactone linker 1230 to an illudin2 moiety 1302 acts as an AMfor the integrin binding peptide receptor and directs the illudin2moiety 1302 to tumors containing cells expressing the receptor. In anembodiment of the present invention, an integrin binding peptide 1150linked azlactone linker 1230 to an illudin2 moiety 1302 acts as an AMfor the integrin binding peptide receptor and directs the illudin2moiety 1302 to tumors containing cells over-expressing the receptorcompared to non tumor cells.

Azlactone linker to bind Illudin2 to a Pro-peptide. In an embodiment ofthe present invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a pro-peptide 1160 is cleaved by an enzyme 1160 andthereafter binds to receptors for the peptide and directs the illudin2moiety 1302 to cell populations expressing the receptor. In anembodiment of the present invention, a pro-peptide linked via anazlactone linker 1230 to an illudin2 moiety 1302 is cleaved by an enzyme1160 and thereafter the peptide acts as an AM for the peptide receptorand directs the illudin2 moiety 1302 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, apeptide linked via an azlactone linker 1230 to an illudin2 moiety 1302is cleaved by an enzyme 1160 and thereafter the peptide directs theilludin2 moiety 1302 to tumors containing cells expressing the receptor.In an embodiment of the present invention, a peptide linked via anazlactone linker 1230 to an illudin2 moiety 1302 is cleaved by an enzyme1160 and thereafter the peptide directs the illudin2 moiety 1302 totumors containing cells over-expressing the receptor compared to nontumor cells.

Azlactone linker to bind Illudin2 to a Glycopeptide. In an embodiment ofthe present invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a glycopeptide 1170 with biological activity binds toreceptors for the glycopeptide 1170 and directs the illudin2 moiety 1302to cell populations expressing the receptor. In an embodiment of thepresent invention, a glycopeptide 1170 linked via an azlactone linker1230 to an illudin2 moiety 1302 acts as an AM for the glycopeptidereceptor and directs the illudin2 moiety 1302 to tissues containingcells expressing the receptor. In an embodiment of the presentinvention, a glycopeptide 1170 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the glycopeptide receptor anddirects the illudin2 moiety 1302 to tumors containing cells expressingthe receptor. In an embodiment of the present invention, a glycopeptide1170 linked via an azlactone linker 1230 to an illudin2 moiety 1302 actsas an AM for the glycopeptide receptor and directs the illudin2 moiety1302 to tumors containing cells over-expressing the receptor compared tonon tumor cells

Azlactone linker to bind Illudin2 to a Lipid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a lipid 1180 with biological activity binds to receptorsfor the lipid 1180 and directs the illudin2 moiety 1302 to cellpopulations expressing the lipid. In an embodiment of the presentinvention, a lipid 1180 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the lipid receptor and directsthe illudin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a lipid 1180 linkedvia an azlactone linker 1230 to an illudin2 moiety 1302 acts as an AMfor the lipid receptor and directs the illudin2 moiety 1302 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

Azlactone linker to bind Illudin2 to a Peptide. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a peptide 1190 with biological activity binds to thepeptide receptor and directs the illudin2 moiety 1302 to cellpopulations expressing the receptor. In an embodiment of the presentinvention, a peptide 1190 linked via an azlactone linker 1230 to anilludin2 moiety 1302 acts as an AM for the peptide receptor and directsthe illudin2 moiety 1302 to tissues containing cells expressing thereceptor. In an embodiment of the present invention, a peptide 1190linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the peptide receptor and directs the illudin2 moiety 1302 totumors containing cells expressing the receptor. In an embodiment of thepresent invention, a peptide 1190 linked via an azlactone linker 1230 toan illudin2 moiety 1302 acts as an AM for the peptide receptor anddirects the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind Illudin2 to a Steroid. In an embodiment of thepresent invention, an illudin2 moiety 1302 linked via an azlactonelinker 1230 to a steroid 1140 binds to receptors for the steroid anddirects the illudin2 moiety 1302 to cell populations expressing thereceptor. In an embodiment of the present invention, a steroid 1140linked via an azlactone linker 1230 to an illudin2 moiety 1302 acts asan AM for the steroid hormone receptor and directs the illudin2 moiety1302 to tissues containing cells expressing the receptor. In anembodiment of the present invention, a steroid 1140 linked via anazlactone linker 1230 to an illudin2 moiety 1302 acts as an AM for thesteroid hormone receptor and directs the illudin2 moiety 1302 to tumorscontaining cells expressing the receptor. In an embodiment of thepresent invention, a steroid 1140 linked via an azlactone linker 1230 toan illudin2 moiety 1302 acts as an AM for the steroid hormone receptorand directs the illudin2 moiety 1302 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Antibody to a Protein Toxin. In anembodiment of the present invention, an antibody 1110 is bound to aprotein toxin 1330 with an azlactone linker 1230. In an embodiment ofthe present invention, an antibody 1110 directs the protein toxin 1330to cell populations expressing the receptor. In an embodiment of thepresent invention, the antibody 1110 with an azlactone linker 1230 to aprotein toxin 1330 acts as an AM for a receptor and directs the proteintoxin 1330 to tissues containing cells expressing the receptor. In anembodiment of the present invention, an antibody 1110 with an azlactonelinker 1230 to the protein toxin 1330 acts as an AM for a receptor anddirects the protein toxin 1330 to tumors containing cells expressing thereceptor. In an embodiment of the present invention, an antibody 1110with an azlactone linker 1230 to the protein toxin 1330 acts as an AMfor a receptor and directs the protein toxin 1330 to tumors containingcells over-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Growth Factor to a Protein Toxin. In anembodiment of the present invention, a growth factor 1120 is bound to aprotein toxin 1330 with an azlactone linker 1230. In an embodiment ofthe present invention, the growth factor 1120 directs the protein toxin1330 to cell populations expressing the receptor to the growth factor1120. In an embodiment of the present invention, the growth factor 1120with an azlactone linker 1230 to a protein toxin 1330 acts as an AM fora receptor to the growth factor 1120 and directs the protein toxin 1330to tissues containing cells expressing the receptor. In an embodiment ofthe present invention, the growth factor 1120 with an azlactone linker1230 to the protein toxin 1330 acts as an AM for the growth factorreceptor and directs the protein toxin 1330 to tumors containing cellsover-expressing the receptor compared to non tumor cells.

Azlactone linker to bind an Antibody to a Medicant. In an embodiment ofthe present invention, an antibody 1110 is bound to a medicant 1350 withan azlactone linker 1230. In an embodiment of the present invention, anantibody 1110 directs the medicant 1350 to cell populations expressingthe receptor. In an embodiment of the present invention, the antibody1110 with an azlactone linker 1230 to the medicant 1350 acts as an AMfor a receptor and directs the medicant 1350 to tissues containing cellsexpressing the receptor. In an embodiment of the present invention, anantibody 1110 with an azlactone linker 1230 to the medicant 1350 acts asan AM for a receptor and directs the medicant 1350 to tumors containingcells expressing the receptor. In an embodiment of the presentinvention, an antibody 1110 with an azlactone linker 1230 to themedicant 1350 acts as an AM for a receptor and directs the medicant 1350to tumors containing cells over-expressing the receptor compared to nontumor cells.

Azlactone linker to bind an Growth Factor to a Medicant. In anembodiment of the present invention, a growth factor 1120 is bound to amedicant 1350 with an azlactone linker 1230. In an embodiment of thepresent invention, the growth factor 1120 directs the medicant 1350 tocell populations expressing the receptor to the growth factor 1120. Inan embodiment of the present invention, the growth factor 1120 with anazlactone linker 1230 to a medicant 1350 acts as an AM for a receptor tothe growth factor 1120 and directs the medicant 1350 to tissuescontaining cells expressing the receptor. In an embodiment of thepresent invention, the growth factor 1120 with an azlactone linker 1230to the medicant 1350 acts as an AM for a receptor and directs themedicant 1350 to tumors containing cells expressing the receptor for thegrowth factor 1120. In an embodiment of the present invention, thegrowth factor 1120 with an azlactone linker 1230 to the medicant 1350acts as an AM for a receptor and directs the medicant 1350 to tumorscontaining cells over-expressing the receptor compared to non tumorcells.

EXAMPLE 16 Synthesis of Medicant 114

(CH₃)₃S(O)I (110 mg, 0.4 mmol) and tBuOK(50 mg, 0.4 mmol) were dissolvedin anhydrous DMSO (1 mL) and stirred at room temperature for 40 minutesat room temperature. Then analog 10 (FIG. 20AJ) (50 mg, 0.2 mmol) in 1.0mL of DMSO was added via syringe, and stirred for 3 hours. Reactionquenched with saturated NH₄Cl (1 mL), extracted with CH₂Cl₂, dried overNa₂SO₄, concentrated then chromatographed (2:3 hexane:ethyl acetate) toyield analog 114 (FIG. 20EJ) (20 mg., 50% yield).

EXAMPLE 17 Synthesis of Medicant 115

Analog 10 (FIG. 20AJ) (40 mg) and NAHCO₃ (50 mg) are dissolved in 10 mLof 1:1 Ethanol and water mixture, then hydroxylamine hydrochloride (20mg) is added, stirred for 30 minutes at room temperature. Water andethyl acetate (1:1 mixture) is added, stirred, the organic layer isrecovered, washed with saturated NaHCO₃ and then brine, dried overNa₂SO₄, concentrated then chromatographed (2:3 ethyl acetate:hexane) toyield analog 115 (FIG. 20EK).

EXAMPLE 18 Synthesis of Medicant 116

SeO₂ (45 mg) and 500 mg SiO2 transferred into a dried RB flask, 5 mL ofCH₂Cl₂ added, and stirred for 1 hour under nitrogen. Then 250 μL oftBuO₂H added and stirred for 15 minutes. Then 100 mg of Irofulven in 1mL CH₂Cl₂ is added, and stirred for 3 hours at room temperature under anitrogen atmosphere. Product is filtered, wash twice with water (25 mL),twice with brine (25 mL), dried over Na₂SO₄ and concentrated thenchromatographed (4:1 hexane:ethyl acetate) to yield analog 116 (FIG.20EL).

EXAMPLE 19 Synthesis of Medicant 116

Analog 117: Illudin S (100 mg, 0.378 mmol) and glutaric anhydride(215.46 mg, 1.89 mmol) are dissolved in 5 mL of CH₂Cl₂, and DMAP added(92.23 mg, 0.756 mmol), and stirred for 2 hours at room temperature. TheCH₂Cl₂ is evaporated, 5 mL of water is added, and stirred for 1 hour.The solution is extracted with 10 mL of CH₂Cl₂, washed with water, driedover Na₂SO₄ and concentrated to yield analog 117 (FIG. 20EM) (120 mg).

EXAMPLE 20 Synthesis of Analog 118

Analog 302 (75 mg), glutaric anhydride (20 mg) are dissolved in 5 mL ofCH₂Cl₂, and DMAP added (42 mg), and stirred for 2 hours at roomtemperature. The CH₂Cl₂ is evaporated, 5 mL of water added, and stirredfor 1 hour. Solution is extracted with 10 mL of CH₂Cl₂, washed withwater, dried over Na₂SO₄ and concentrated to yield analog 118 (120 mg)(FIG. 20EN).

EXAMPLE 21 Synthesis of Analog 119

Analog 114 (10 mg) is dissolved in 1.5 mL of acetone with 1.0 mL of 4NH₂SO₄, and contents stirred for 1.5 hours at room temperature. Then 10mL of CH₂Cl₂ and 10 mL of water are added, extracted, and the organiclayer recovered which is then washed with saturated NaHCO₃ and saline,dried over Na₂SO₄ and concentrated, and analog 119 recovered (analog 128is a byproduct).

EXAMPLE 22 Synthesis of Analog 120

Analog 10 (50 mg), NaHCO₃ are dissolved in 10 mL of 1:1 mixture of waterand ethanol, then NH₂NH₂ (0.5 mL added with stirring at room temperaturefor one hour. The solution is extracted with CH₂Cl₂ twice, the organiclayer recovered, washed with water, then NaHCO₃ solution, dried overNa₂SO₄, and evaporated to yield analog 120 (30 mg).

EXAMPLE 23 Synthesis of Analog 121

Analog 10 (50 mg) and NaCO₂CH₃ (75 mg) are dissolved in 10 mL of 1:1mixture of water and ethanol 1:1, then semicarbazide hydrochloride salt(H₂NNHCONH₂HCl, 50 mg) added, and stirred for 2 hours at roomtemperature. The solution is extracted with CH₂Cl₂ twice, the organiclayer recovered, washed with water, then NaHCO₃ solution, dried overNa₂SO₄, and evaporated then chromatographed (5% methanol in ethylacetate) to yield analog 121.

EXAMPLE 24 Synthesis of Analog 122

Analog 10 (50 mg) and NaCO₂CH₃ (75 mg) are dissolved in 5 mL of ethanol,then phenylhydrazide (50 mg) is added, stirred for 1 hour at roomtemperature. Then 5 mL of water is added, followed by extraction withethyl acetate, washed with water, dried over Na₂SO₄ and concentrated andchromatographed (5% methanol in ethyl acetate) to yield analog 122.

EXAMPLE 25 Synthesis of Analog 123

Analog 10 (50 mg) and NaCO₂CH₃ (75 mg) are dissolved in 10 mL of 1:1water and ethanol, then H₂NNHTS (H₂NNHS(═O)₂(phenyl)methyl, 50 mg) isadded, stirred for 2 hour at room temperature. Then 5 mL of water isadded, followed by extraction with ethyl acetate, washed with water,dried over Na₂SO₄ and concentrated and chromatographed (5% methanol inethyl acetate) to yield analog 123.

EXAMPLE 26 Synthesis of Analog 124

Analog 115 (15 mg) and NaOAc(15 mg) are dissolved in acetic anhydride (1mL) and stirred for 2 hours, then sodium acetate (300 mg) is added withstirring for 1 hour. Then the mixture is chromatographed (10% ethylacetate in hexane) to give analog 124.

EXAMPLE 27 Synthesis of Analog 125

Analog 10 (50 mg) and NaCO₂CH₃ (75 mg) are dissolved in 5 mL of ethanol,then the dinitrophenylhydrazine (50 mg) is added, stirred for 1 hour atroom temperature. Then 5 mL of water is added, followed by extractionwith ethyl acetate, washed with water, dried over Na₂SO₄ andconcentrated and chromatographed (5% methanol in ethyl acetate) to yieldanalog 125.

EXAMPLE 28 Synthesis of Analog 126

Analog 11 (40 mg), hydroxylamine (20 mg), NaHCO₃ (50 mg) are dissolvedin 10 mL of ethanol and water (1:1) then stirred at room temperature for90 minutes. Then the mixture is extracted with water (10 mL) and ethylacetate (20 mL), the organic layer washed with saturated NaHCO₃ thenbrine, dried over Na₂SO₄ and concentrated, then chromatographed (2:3ethyl acetate:hexane) to give analog 126.

EXAMPLE 29 Synthesis of Analog 127

Analog 10 (100 mg) and NH₄Cl (1.5 equivalent) are dissolved in1,4-dioxane (5 mL) and water (0.2 mL), then NaCN added (1.3equivalents), stirred for 1 hour at room temperature. Then ethyl ether(20 mL) was added, the organic layer recovered, washed with water,washed with brine, then dried over Na₂SO₄, then chromatographed (2:3ethyl acetate: hexane) to yield analog 127.

EXAMPLE 30 Synthesis of Analog 128

Analog 114 (10 mg) is dissolved in 1.5 mL of acetone with 1.0 mL of 4NH₂SO₄, and contents stirred for 1.5 hours at room temperature. Then 10mL of CH₂Cl₂ and 10 mL of water are added, extracted, and the organiclayer recovered which is then washed with saturated NaHCO₃ and saline,dried over Na₂SO₄ and concentrated, and analog 128 recovered (analog 119is a byproduct).

EXAMPLE 31 Synthesis of Analog 129

Acylfulvene (200 mg) is dissolved in anhydrous THF (10 mL) at roomtemperature then NaBH₄ (100 mg) is added slowly for 30 minutes. Reactionis quenched with 1 mL of water then extracted with ethyl acetate (10mL), washed with saturated NaHCO₃, and dried over Na₂SO₄, thenconcentrated to yield analog 129. If need be the compound can bepurified by chromatography (1:1 ethyl acetate: hexane).

EXAMPLE 32 Analog 141

Analog 129 (200 mg) is dissolved in CH₂Cl₂ at room temperature, then1,4-dimethyl but-2-ynedioate (1.1 equivalent) is added slowly andmixture allowed to read for one hour, then evaporated to yield analog141. If need be the compound can be purified by chromatography (1:1ethyl acetate: hexane).

EXAMPLE 33 Synthesis of Analog 142

Analog 141 (100 mg) is dissolved in CH₂Cl₂ at room temperature thenDess-Martin Periodinane reagent (200 mg) added with stirring for 1 hourto yield analog 142. If need be the compound can be purified bychromatography (1:1 ethyl acetate: hexane).

EXAMPLE 34 Synthesis of Analog 146

Analog 127 (35 mg, 0.117 mmol), DMAP (5 mg), and diimidazole (22 mg, 1.2eq) were dissolved in anhydrous CH₂Cl₂ under an argon atmosphere, andstirred for 30 minutes. The solution was cooled to 20° C. then tributyltin hydride (Bu₃SnH, 0.6 mL) and azobis isobutylnitrite (4 mg) wereadded with stirring for 30 minutes. The mixture was filtered thenchromatographed (1:10 ethyl acetate: hexane) to remove impurities andstarting materials, then chromatographed (2:3 ethyl acetate: hexane) toyield analog 146.

EXAMPLE 35 Synthesis of Analog 147

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and 2-Mercaptobenzothiazole (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 147.

EXAMPLE 36 Synthesis of Analog 148

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and 2-Mercaptobenzoxazole (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 Ethyl acetate: hexane) to give analog 148.

EXAMPLE 37 Synthesis of Analog 149

Irofulven (10 mg) is dissolved in 4 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and thiol-imidazole (1equivalent) is added, stirred for 24 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 149.

EXAMPLE 38 Synthesis of Analog 150

Irofulven (10 mg) is dissolved in 4 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and2-mercapto-5-methylbenzimidazole (1 equivalent) is added, stirred for 12hours, then partitioned between ethyl acetate and water. The organicextract is washed with saturated NaHCO₃ and saline until neutral, driedover MgSO₄, concentrated then chromatographed (1:1 ethyl acetate:hexane)to give analog 150.

EXAMPLE 39 Synthesis of Analog 151

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and1-phenyl-1,2,3,4-tetraazole-5-thiol (1 equivalent) is added, stirred for2 hours, then partitioned between ethyl acetate and water. The organicextract is washed with saturated NaHCO₃ and saline until neutral, driedover MgSO₄, concentrated then chromatographed (1:1 ethyl acetate:hexane)to give analog 151.

EXAMPLE 40 Synthesis of Analog 152

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and 2-mercapto-5-nitrobenzimidazole (1 equivalent) is added, stirred for 2 hours, thenpartitioned between ethyl acetate and water. The organic extract iswashed with saturated NaHCO₃ and saline until neutral, dried over MgSO₄,concentrated then chromatographed (1:1 ethyl acetate:hexane) to giveanalog 152.

EXAMPLE 41 Synthesis of Analog 153

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and 1,2,4-Triazole-3-thiol (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 153.

EXAMPLE 42 Synthesis of Analog 154

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and 2-sulfanylpteridin-4-ol (1equivalent) is added, stirred for 2 hours, then partitioned betweenethyl acetate and water. The organic extract is washed with saturatedNaHCO₃ and saline until neutral, dried over MgSO₄, concentrated thenchromatographed (1:1 ethyl acetate:hexane) to give analog 154.

EXAMPLE 43 Synthesis of Analog 155

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and4-(5-sulfanyl-1H-1,2,3,4-tetrazol-1-yl)phenol (1 equivalent) is added,stirred for 2 hours, then partitioned between ethyl acetate and water.The organic extract is washed with saturated NaHCO₃ and saline untilneutral, dried over MgSO₄, concentrated then chromatographed (1:1 ethylacetate:hexane) to give analog 155.

EXAMPLE 44 Synthesis of Analog 156

Irofulven (10 mg) is dissolved in 3 mL of acetone and 1 M H₂SO₄ solution(1:1) with stirring at room temperature and4-(5-sulfanyl-1-1,2,3,4-tetrazol-1-yl)benzoic acid (1 equivalent) isadded, stirred for 2 hours, then partitioned between ethyl acetate andwater. The organic extract is washed with saturated NaHCO₃ and salineuntil neutral, dried over MgSO₄, concentrated then chromatographed (1:1ethyl acetate:hexane) to give analog 156.

EXAMPLE 45 Synthesis of Analog 159

Illudin S (300 mg) is dissolved acetic anhydride (6 mL) and stirred for15 minutes, then sodium acetate (300 mg) is added with stirring for 1hour. Water (6 mL) is added, ethyl acetate extraction performed, washedwith sodium bicarbonate solution, dried over Na₂SO₄, concentrated thenchromatographed (2:3 ethyl acetate:hexane) to give analog 159.

EXAMPLE 46 Synthesis of Analog 160

Analog 159 (60 mg) is dissolved in dry CH₂Cl₂ (6 mL) under nitrogen atroom temperature and glutaric anhydride (100 mg) with DMAP (20 mg) isadded with stirring for 30 minutes. The solvent is removed, water added,extracted with CH₂Cl₂, washed with water, dried over Na₂SO₄,concentrated then chromatographed (2:3 ethyl acetate: hexane) to giveanalog 160.

EXAMPLE 47 Synthesis of Analog 161

Dehydroilludin S (300 mg) is dissolved acetic anhydride (6 mL) andstirred for 15 minutes, then sodium acetate (300 mg) is added withstirring for 1 hour. Water (6 mL) is added, ethyl acetate extractionperformed, washed with sodium bicarbonate solution, dried over Na₂SO₄,concentrated then chromatographed (2:3 ethyl acetate:hexane) to giveanalog 161.

EXAMPLE 48 Synthesis of Analog 162

Dehydroilludin S (60 mg) is dissolved in dry CH₂Cl₂ (6 mL) undernitrogen at room temperature and glutaric anhydride (150 mg) with DMAP(50 mg) is added with stirring for 30 minutes. The solvent is removed,water added, extracted with CH₂Cl₂, washed with water, dried overNa₂SO₄, concentrated then chromatographed (2:3 ethyl acetate:hexane) togive analog 162.

EXAMPLE 49 Synthesis of Analog 163

Analog 159 (20.25 mg), DMAP (20 mg) are dissolved in dry CH₂Cl₂ (6 mL)at 0° C. under nitrogen atmosphere and stirred for 10 minutes. Thenchloroacetyl chloride (0.2 mL) is added slowly and the mixture stirredfor 30 minutes, warmed to room temperature with stiffing over 1.5minutes. Then water (6 mL) is added, mixed, and then extracted withCH₂Cl₂. The organic layer is washed with saturated NaHCO₃ followed by asaline wash, dried over Na₂SO₄ then chromatographed (2:3 ethyl acetate:hexane) to yield analog 163 (60% yield).

EXAMPLE 50 Synthesis of Analog 164

Irofulven (50 mg), DMAP (40 mg) are dissolved in dry CH₂Cl₂ (6 mL) at 0°C. under nitrogen atmosphere and stirred for 10 minutes. Thenchloroacetyl chloride (0.2 mL) is added slowly and the mixture stirredfor 30 minutes, warmed to room temperature with stiffing over 1.5minutes. Then water (6 mL) is added, mixed, and then extracted withCH₂Cl₂. The organic layer is washed with saturated NaHCO₃ followed by asaline wash, dried over Na₂SO₄ then chromatographed (2:3 ethyl acetate:hexane) to yield analog 164 (60% yield).

EXAMPLE 51 Synthesis of Analog 165

Analog 164 (40 mg) is dissolved in dry CH₂Cl₂ (6 mL) at room temperatureunder nitrogen atmosphere and stirred for 10 minutes. Then 1 mL ofmorpholine is added drop wise, with stiffing for 30 minutes. Thereaction is diluted with water (6 mL), extracted with CH₂Cl₂ (12 mL).The organic layer is washed with saturated NaHCO₃ then washed withsaline, dried over Na₂SO₄ and chromatographed (2:3 ethyl acetate:hexane) to yield 165 (35% yield).

EXAMPLE 52 Synthesis of Analog 166 and Analog 167 (Prepared Together)

Analog 160 (30 mg) is dissolved in methanol (4 mL) at 0° C., and 1NH₂SO₄ (1 mL) is added with stiffing for 1 hour. Water (6 mL) is added,extracted with ethyl acetate, washed with NaHCO₃ then a brine solution,dried over MgSO₄, concentrated and then chromatographed (1:1 ethylacetate:hexane) to yield analogs 166 and 167 in equal amounts.

EXAMPLE 53 Synthesis of Analog 168

Analog 162 (20 mg) is dissolved in methanol (5 mL) at 0° C. and stirredfor 10 minutes, then 1 mL of 1N H₂SO₄ in methanol is slowly added,followed by stirring for 30 minutes. Water is added, followed by anethyl acetate extraction, washed with NaHCO₃ then a brine solution,dried over Na₂SO₄, concentrated then chromatographed (1:1 ethylacetate:hexane) to yield analog 168.

EXAMPLE 54 Synthesis of Analog 169

Dehydroilludin S (20 mg), DMAP (20 mg) are dissolved in dry CH₂Cl₂ (6mL) at 0° C. under nitrogen atmosphere and stirred for 10 minutes. Thenchloroacetyl chloride (0.2 mL) is added slowly and the mixture stirredfor 30 minutes, warmed to room temperature with stiffing over 15minutes. Then water (6 mL) is added, mixed, then extracted with CH₂Cl₂.The organic layer is washed with saturated NaHCO₃ followed by a salinewash, dried over Na₂SO₄ then chromatographed (2:3 ethyl acetate: hexane)to yield analog 169 (60% yield).

EXAMPLE 55 Synthesis of Analog 176

To a solution of analog 9 (266 umol), Boc protected leucine amino acid(300 umol) and DMAP (dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL)at 0° C. is added DCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300umol)/. The mixture is stirred for 35 minutes then 5 μL of water addedto quench the reaction. The mixture is diluted with hexane andprecipitate filtered off, solvent evaporated off and crude productchromatographed (2:1 hexanes-ethyl acetate) to give the desiredBoc-protected derivative of 176 at 80% yield. The Boc group is removedby dissolving the Boc-protected derivative in a 1:1 mixture (2.0 mL) of1,4-dioxane and 2M H₂SO₄, stirred for 18 hours, then partitioned betweenethyl acetate and water. Aqueous layer is extracted with ethyl acetateand extracts discarded. Aqueous layer is neutralized with saturatedNaHCO₃ and extracted again with ethyl acetate. Organic layer is washedwith brine, dried with MgSO₃, solvent evaporated to yield the analog 9amino acid derivative. As the amine derivative is unstable overprolonged periods of time it can be converted to the very stabletrifluoroacetate salt by dissolving in CH₂Cl₂ adding the equal molaramount of trifluoroacetic acid and concentrating to dryness.

EXAMPLE 56 Synthesis of Analog 178

Analog 9 (15 mg) is dissolved in CH₂Cl₂ (2.0 mL) under a nitrogenatmosphere at room temperature, glutaric anhydride (1 equivalent) isadded, followed by DMAP (10 mg) and stirring for 30 minutes. Solvent isremoved and product recrystallized to give analog 178.

EXAMPLE 57 Synthesis of Analog 179

To a solution of Analog 9 (266 μmol), Boc protected glycine amino acid(300 umol) and DMAP (dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL)at 0° C. is added DCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300umol)/. The mixture is stirred for 35 minutes then 5 μL of water addedto quench the reaction. The mixture is diluted with hexane andprecipitate filtered off, solvent evaporated off and crude productchromatographed (2:1 hexanes-ethyl acetate) to give the desiredBoc-protected derivative of 179 at 80% yield. The Boc group is removedby dissolving the Boc-protected derivative in a 1:1 mixture (2.0 mL) of1,4-dioxane and 2M H₂SO₄, stirred for 18 hours, then partitioned betweenethyl acetate and water. Aqueous layer is extracted with ethyl acetateand extracts discarded. Aqueous layer is neutralized with saturatedNaHCO₃ and extracted again with ethyl acetate. Organic layer is washedwith brine, dried with MgSO₃, solvent evaporated to yield the analog 9amino acid derivative. As the amine derivative is unstable overprolonged periods of time it can be converted to the very stabletrifluoroacetate salt by dissolving in CH₂Cl₂ adding the equal molaramount of trifluoroacetic acid and concentrating to dryness.

EXAMPLE 58 Synthesis of Analog 180

Illudin M (50 mg) is dissolved in dry benzene (10 mL) under a nitrogenatmosphere, and vanadyl acetylacetonate (VO(acac)₂, 1.2 mg) is added.Then t-butyl hydroperoxide (t-BuO₂H, 0.5 mL) in benzene is added dropwise with stirring for 30 minutes. A saturated solution of Na₂S₂O₃ isadded (10 mL), then extraction with ethyl acetate, and the organic layeris dried over Na2SO₄, concentrated then chromatographed) (1:1 ethylacetate:hexane) to give analog 180.

EXAMPLE 59 Synthesis of Analog 181

Analog 159 (40 mg) was dissolved in dry benzene (8 mL) under a nitrogenatmosphere, and vanadyl acetylacetonate (VO(acac)₂, 2 mg) was added.Then t-butyl hydroperoxide (t-BuO₂H, 0.5 mL) in benzene was added dropwise with stirring for 30 minutes. A saturated solution of Na₂S₂O₃ isadded (10 mL), then extraction with ethyl acetate, followed by a brinewash, and the organic layer was then dried over Na₂SO₄, concentratedthen chromatographed) (1:1 ethyl acetate:hexane) to give analog 181.

EXAMPLE 60 Synthesis of Analog 189

To a solution of Irofulven (1.00 equivalent), maleimide (1.71equivalent), triphenylphosphine (PPh₃, 1.71 equivalent) in 1.5 mL of THFat −40° C., is added DEAD (diethylazodicarboxylate; 1.68 equivalent).The mixture is stirred for 30 minutes then water (20 μL) added to quenchthe reaction. The mixture is concentrated on a rotary evaporator andcrude product is chromatographed on a silica column (10:3 hexanes: ethylacetate) to yield an orange compound (20% yield).

EXAMPLE 61 Synthesis of Analog 190

To a solution of analog 9 (6-hydroxy-n-propylacylfulvene—structurebelow, 1.00 equivalent), maleimide (1.23 equivalent), triphenylphosphine(PPh₃, 1.13 equivalent) in 2.5 mL of THF at −40° C., is added DIAD(diisopropylcarbodiimide; 1.44 equivalent). The mixture is stirred for 1hour then water (10 μL) added to quench the reaction. The mixture isconcentrated on a rotary evaporator and crude product is chromatographedon a silica column (5:1→10:3 hexanes:ethyl acetate) to yield an orangecompound (15% yield).

EXAMPLE 62 Synthesis of Analog 196

To a solution of analog 9 (266 umol), Boc protected proline amino acid(300 umol) and DMAP (dimethylaminopyridine, 110 umol) in CH₂Cl₂ (2.5 mL)at 0° C. is added DCC (dicyclohexylcarbodiimide; 1.0M in CH₂Cl₂, 300umol)/. The mixture is stirred for 35 minutes then 5 μL of water addedto quench the reaction. The mixture is diluted with hexane andprecipitate filtered off, solvent evaporated off and crude productchromatographed (2:1 hexanes-ethyl acetate) to give the desiredBoc-protected derivative of 196 at 80% yield. The Boc group is removedby dissolving the Boc-protected derivative in a 1:1 mixture (2.0 mL) of1,4-dioxane and 2M H₂SO₄, stirred for 18 hours, then partitioned betweenethyl acetate and water. Aqueous layer is extracted with ethyl acetateand extracts discarded. Aqueous layer is neutralized with saturatedNaHCO₃ and extracted again with ethyl acetate. Organic layer is washedwith brine, dried with MgSO₃, solvent evaporated to yield the analog 9amino acid derivative. As the amine derivative is unstable overprolonged periods of time it can be converted to the very stabletrifluoroacetate salt by dissolving in CH₂Cl₂ adding the equal molaramount of trifluoroacetic acid and concentrating to dryness.

EXAMPLE 63 Synthesis of Analog 198

Irofulven (26.3 mg, 107 umol), p-nitrophenol (16.2 mg, 116 umol) andPPh3 (30.8 mg, 117 umol) were dissolved in anhydrous THF (1.5 mL) at−40° C., the DEAD (25 μL, 160 umol) was added, followed by stiffing for30 minutes, then diluted with hexane. The precipitate was filtered off,solvent evaporated, and crude product chromatographed (6:1→2:1 hexane:ethyl acetate) to give analog 198 (FIG. 20HO) as a yellow product (18.5mg, 47%).

EXAMPLE 64 Analogs 199 and 200 (Prepared Together)

Irofulven (25.2 mg, 102 umol), phenol (11.5 mg, 122 umol) and PPh₃ (29.1mg, 117 μmol) were dissolved in anhydrous THF (1.0 mL) at −40° C., theDEAD (25 μL, 192 μmol) was added, followed by stiffing for 30 minutes,then diluted with hexane. The precipitate was filtered off, solventevaporated, and crude product chromatographed (6:1→3:1 hexane: ethylacetate) to give analog 199 (FIG. 20HP) (8.2 mg, 25%) and analog 200(FIG. 20HQ) (14.6 mg, 44%) as a yellow products.

EXAMPLE 65 Synthesis of Analog 201 [6-(acetamidopropyl)acylfulvene]

To a solution of analog 195 (FIG. 20HL) (49.1 umol) and water (20 μL inTHF (0.5 ml) was added a solution ofO-acetyl-2-(diphenylphosphino)phenol (39.0 umol) in THF (0.5 mL). Themixture was stirred for 3 days at room temperature then concentrated.The crude product was chromatographed (100% ethyl acetate) to yield 8.2mg of analog 201 (FIG. 20HR).

EXAMPLE 66 Synthesis of Analog 202

((FIG. 20HS, i.e., analog 211 linked to proline): Prepared viaStaudinger ligation. To a solution of analog 195 (FIG. 20HL) (94 umol)in THF (1.2 mL), water (40 μL) was added, the was added N-Boc-proline,2-(diphenylphosphino)phenyl ester (101 μmol) in THF (0.8 mL). Themixture was stirred for 3 days at room temperature then concentrated.The crude product was chromatographed (5:1->1:2 hexanes-ethyl acetate)to yield 31.4 mg (66.7 umol) of analog 202 (FIG. 20HS) -Boc (71%). Theanalog 202-Boc was dissolved (66.7 umol) in dioxane (2.0 mL) and 2.0 mLof 2M H₂SO₄ was added, and the mixture was stirred overnight. Water andethyl acetate was added, orange color appeared in the aqueous. Theaqueous was extracted again with ethyl acetate and organic layerdiscarded. Sodium bicarbonate was added to aqueous until basic,re-extracted with ethyl acetate. The solution was dried with magnesiumsulphate, concentrated to dryness, dissolved in CH₂Cl₂ and 8 mg of TFAadded (1 drop). Analog 202 (FIG. 20HS) was obtained in an amount of 22.2mg (69%).

EXAMPLE 67 Synthesis of Analog 203

Synthesis of Analog 208 (FIG. 20HY) (9.2 mg, 16.5 umol) is dissolved inCH₂Cl₂ (1.5 mL), 1 drop of anisole added, then 0.5 mL of trifluro aceticacid for 15 minutes. The mixture is concentrated, dissolved in water,then re-extracted with CH₂Cl₂, and the orange color remains in theaqueous phase, which is concentrated to give analog 203 (FIG. 20HT) asthe orange colored TFA salt (10.0 mg).

EXAMPLE 68 Synthesis of Analog 204

Although the Fmoc-Pro-OH would preferentially react with the primaryhydroxyl group on Illudin S, the resulting ester linkage is not stable,as illudin S was recovered after storage in CDCl₃ for several days atroom temperature. The secondary hydroxy group of illudin S was thereforeused for coupling with peptides. The primary hydroxy group of illudin Sfirst protected with a TBS group (TBSC1, Imidazole, and DMF, 92%) toproduce analog 204 (FIG. 20HU).

EXAMPLE 69 Synthesis of Analog 205

Analog 309 (FIG. 20LV) (20 mg, 0.050 mmol, 1 equiv.), triphenylphosphine(40 mg, 0.1525 mmol, 3 equiv.) was dissolved in THF (1 mL) at roomtemperature. After 20 hours a few drops of water was added and themixture was heated up at 70° C. After 5 hours the solution was cooleddown and evaporated. The residue was chromatographed (hexane/EtOAc/Et₃N4:1:0.1) to give analog 205 (FIG. 20HV) (5.3 mg, 29%) as an oil.

EXAMPLE 70 Synthesis of Analog 206

Analog 205 (FIG. 20HV) (14 mg, 0.037 mmol, 1 equiv.) was dissolved inCH₃CN (0.5 mL) and pyridine (0.1 mL) at 0° C. To this solution was addedHF.Pyridine (7 μL, 0.245 mmol, 35 M, 6.6 equiv.). After 10 min K₂CO₃(0.5 mL, 0.5 M) was added and this mixture was chromatographed(CH₂Cl₂/Methanol/Et₃N 5:0.5:0.1) to give analog 206 (FIG. 20HW) (10 mg,68%) as an oil.

EXAMPLE 71 Synthesis of Analog_207 (211(FIG. 20IB) -leucine)

Prepared via Staudinger ligation. To a solution of analog 195 (FIG.20HL) (101 umol) in THF (1.0 mL), water (40 μL) was added, then wasadded N-Boc-leucine, 2-(diphenylphosphino)phenyl ester (95.9 μmol) inTHF (1.2 mL). The mixture was stirred for 6 days at room temperaturethen concentrated. The crude product was chromatographed (1:1hexanes-ethyl acetate) to yield 27.3 mg of analog 207 (FIG. 20HX) -Boc.The analog 207-Boc was dissolved (16 μmol) in CH₂Cl₂ with 3 drops ofanisole, TFA was added (0.3 mL), and the mixture was stirred for 15minutes then concentrated. The crude material was dissolved in waterthen extracted with CH₂Cl₂. The aqueous layer was recovered andconcentrated to yield 17.4 mg of the analog 207 (FIG. 20HX) TFA salt(87%).

EXAMPLE 72 Analog 208

The TFA salt of analog 196 (FIG. 20HM) (13.7 mg, 28.2 μmol) wasdissolved in anhydrous DMF (2.5 mL), Boc-Serine-OH (9.6 mg, 47 umol) wasadded, ODHBT (13.0 mg, 79.4 umol), cooled to 0° C. under a nitrogenatmosphere. Next EDC (15.1 mg) was added followed by NMM (10 μL) toadjust pH, and the mixture stirred at 0° C. for 3 hours. The reactionwas added to ethyl acetate/water mixture, and the orange productappeared in the organic layer. The aqueous layer was re-extracted withethyl acetate, organic layers combined, washed with dilute NaHSO₄,water, saturated NaHCO₃, brine, then dried with MgSO₄. The organic layerwas concentrated then chromatographed (1:3 hexane: ethyl acetate) toyield analog 208 (FIG. 20HY) as an orange residue (63% yield).

EXAMPLE 73 Synthesis of Analog 209

The TFA salt of analog 196 (FIG. 20HM) (12.5 mg, 25.7 μmol) wasdissolved in anhydrous DMF (2.5 mL), Boc-Serine-Ser OH (88.6 umol) wasadded, ODHBT (33.9 mg, 205 umol), cooled to 0° C. under a nitrogenatmosphere. Next EDC (142 umol) was added followed by NMM (10 μL) toadjust pH, and the mixture stirred at 0° C. but allowed to graduallywarm as the ice melts. The mixture was stirred a total of 16 hour then 1mL water added followed by stiffing for 50 minutes. The reaction wasadded to ethyl acetate/water mixture, and the orange product appeared inthe organic layer. The aqueous layer was re-extracted with ethylacetate, organic layers combined, washed with dilute NaHSO₄, water,saturated NaHCO₃, brine, and then dried with MgSO₄. The organic layerwas concentrated then chromatographed (10:1 ethyl acetate: methanol) togive analog 209 (FIG. 20HZ) as an orange residue (5.9 mg, 36% yield).

EXAMPLE 74 Synthesis of Analog 210

(Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-O—(CH₂)₃-acylfulvene): To a mixtureof Analog 196 (FIG. 20HL) TFA salt (21.6 umol), the peptideAc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (30.3 umol), ODHBt(3,4,-dihydroxy-4-oxo-1,2,3-benzo-triazine-3-yl ester, 71.7 μmol) andNMM (N-methylmorpholine; 7.5 ul) in DMF (2.0 ml) at room temperature isadded EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,68 μmol), the mixture stirred for 2 hours at room temperature, thendiluted with 10 mL of water. Solution is directly chromatographed on areverse phase C18 column (4:1->2:1, water/acetonitrile gradient) toyield 69% of analog 210 (FIG. 20IA).

EXAMPLE 75 Synthesis of Analog 212

(Illudin M-proline) Illudin M (20 mg, 0.081 mmol, 1 equivalent), DMAP (1mg, 0.008 mmol, 0.1 equiv.) and Fmoc-Pro-OH (33 mg, 0.097 mmol, 1.2equiv.) were dissolved in CH₂Cl₂ (1 mL) at 0° C., to which was added aCH₂Cl₂ solution of DCC (100 μL, 0.1 mmol, 1 M, 1.2 equiv.). Thetemperature of the mixture gradually rose to 5° C. in 1.5 hours and thenthe mixture was filtered through a pad of Celite. The filtrate wasconcentrated and the residue was chromatographed (CH₂Cl₂/EtOAc5:0.1-5:0.4) to give Illudin-M-proline-Fmoc protected analog (36 mg,79%) as oil. The proton spectra of this oil showed that it was a mixtureof two isomers (rotamers). And then this oil was dissolved in CH₂Cl₂ (4mL) and treated with piperidine (1 mL) at 0° C. After 0.5 hours thesolution was concentrated and the concentrate was chromatographed(CH₂Cl₂/Methanol 5:0.4) to give analog 212 (FIG. 20IC) (15 mg, 54%) asoil.

EXAMPLE 76 Synthesis of Analog 213

Analog 204 (FIG. 20HU) is coupled with Fmoc-Pro-H (DMAP, CH₂Cl₂, DCC, 0°C., 85%), followed by deprotection of Fmoc group with 20% piperidine inCH₂Cl₂ to produce analog 213 (FIG. 20ID) in 78% yield.

EXAMPLE 77 Synthesis of Analog 214

(Illudin S-Pro-Ser-Ser-HHOAc): The Fmoc protected peptide ofH-Ser-Ser-OH was prepared by taking H-Ser-Ser-OH (50 mg, 0.26 mmol, 1equiv.) and K₂CO₃ (89.7 mg, 0.65 mmol, 2.5 equiv.), dissolving in amixture of water (4 mL) and dioxane (3 mL) at 0° C. To this solutionFmoCl (67.3 mg, 0.26 mmol, 1 equiv.) was added in several portions.After 18 hours the mixture was acidified by KHSO₄ and the pH raised to2.5. Then this mixture was taken up by ethyl acetate, which was washedwith brine, dried, filtered and evaporated. The residue waschromatographed (CH₂Cl₂/Methanol/HOAc 5:1:0.1) to give 3.27 (75 mg, 70%)as a white solid. The analog 212 (FIG. 20IC) (Illudin S tosylate-Pro)(42.8 mg 0.09 mmol, 0.9 equiv.), and the Fmoc protected H-Ser-Ser-OHpeptide (41.2 mg, 0.1 mmol, 1 equiv.) were dissolved in DMF (1.5 mL) at0° C. To this solution was added NMM (22 μL, 0.2 mmol, 2 equiv.), ODHBt(29.4 mg, 0.18 mmol, 1.8 equiv.), and EDC (31.1 mg, 0.16 mmol, 1.6equiv.). The solution temperature was then raised to room temperatureand kept for 3 hours before it was taken up by ethyl acetate. Themixture was then washed with saturated sodium bicarbonate and brine. Itwas then dried, filtered and evaporated. The residue was chromatographed(CH₂Cl₂/Methanol 5:0.3) to give analog 214 (FIG. 20IE) (50.5 mg, 67%) asan oil.

EXAMPLE 78 Synthesis of Analog 215

(Illudin S-Pro-Ser-Ser-Gln-Chg-Ser-Ser-Hyp-Ac) Analog 204 (FIG. 20HU) iscoupled with Fmoc-Pro-H (DMAP, CH₂Cl₂, DCC, 0° C., 85%), followed bydeprotection of Fmoc group with 20% piperidine in CH₂Cl₂ to produceanalog 213 (FIG. 20ID) in 78% yield. Peptide conjugate, analog 215 (FIG.20IF) was obtained from further coupling with hepta-peptideAc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (ODHBt, NMM, DMF, 0° C., 47%).

EXAMPLE 79 Synthesis of Analog 216

(Illudin M-Pro-Ser-Ser-Gln-Chg-Ser-Ser-Hyp-Ac). Analog 212 was furthercoupled with the commercially available hepta-peptideAc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-OH (ODHBt, NMM, DMF, EDC, 0° C.) to yieldanalog 216 (FIG. 20IG) at 33%. The low yield resulted from repeatedchromatographic purification as the purity of the final raw product wasestimated by HPLC to be only 70%.

EXAMPLE 80 Synthesis of Analog 217

To a solution of Irofulven (1.00 equivalent), epsilon-maleimidocaproicacid (1.27 equivalent), DMAP (0.15 equivalent) in 1.0 mL of methylenechloride (CH₂Cl₂) at 0° C., is added DCC (dicyclohexylcarbodiimide; 1.27equivalent) in methylene chloride (CH₂Cl₂). The mixture is stirred for1.25 hours, diluted with hexane and precipitated is filtered. Residualsolvent is evaporated off, and oil residue is chromatographed on asilica column (2:1 hexanes:ethyl acetate) to yield analog 217 (FIG.20II), an orange compound (77% yield).

EXAMPLE 81 Synthesis of Analog 218

To a solution of Illudin M (1.00 equivalent), epsilon-maleimidocaproicacid (1.33 equivalent), DMAP (0.18 equivalent) in 1.0 mL of methylenechloride (CH₂Cl₂) at 0° C., is added DCC (dicyclohexylcarbodiimide; 1.33equivalent) in methylene chloride (CH₂Cl₂). The mixture is stirred for2.25 hours, diluted with hexane and precipitated is filtered. Residualsolvent is evaporated off, and oil residue is chromatographed on asilica column (2:1 hexane: ethyl acetate) to yield analog 218 (FIG.20II), an orange compound (83% yield).

EXAMPLE 82 Synthesis of Analog 219

Analog 204 (33.4 mg) is dissolved in 1.0 mL of anhydrous pyridine undera nitrogen atmosphere, then DMAP (5.1 mg) is added, followed by4-fluorosulfonyl-benzoyl chloride (86.1 mg). The mixture is stirred for90 minutes at room temperature The mixture is diluted with ethylacetate, washed once with saturated copper sulfate solution, washedtwice with water, then dried over MgSO₄, concentrated thenchromatographed (20% ethyl acetate: hexane) to give analog 219 (FIG.20IJ).

EXAMPLE 83 Synthesis of Analog 221

Prepared from Analog 207 by coupling withMu-His-Ser-Ser-Lys(Fmoc)-Leu-Gln-OH in DIC/HOBt for 5 minutes, then 5%piperidine/DMF for 1 minute. Followed by TFA quenching to yield analog221 (FIG. 20IL) at 21% yield.

EXAMPLE 84 Synthesis of Analog 222

Illudin M (63 mg) is dissolved in 1.0 mL of anhydrous pyridine under anitrogen atmosphere, then DMAP (6.4 mg) is added, followed by4-fluorosulfonyl-benzoyl chloride (86 mg). The mixture is stirred for 35minutes at room temperature then chromatographed (20% ethyl acetate:hexane) to give analog 222 (FIG. 20IM) (70.9 mg).

EXAMPLE 85 Synthesis of Analog 223

The disulfhydryl peptide CNGRC is first converted to a cyclic disulfidepeptide by dissolving 355 mg in 3.0 mL DMSO, adding 9 mL of water,allowing to sit overnight at room temperature, followed by water removalon a rotoevaporator then DMSO removal under high vacuum. The TFA salt ofanalog 179 (FIG. 20GV) (14.5 mg) is dissolved in DMF (2.0 mL) and theCNGRC disulfide peptide added (19.0 mg), 60 μL of DIPEA is added,followed by gradual addition of a solution of Py-BOP (19.6 mg) and HOBt(8.9 mg) in DMF (2.0 mL) over 150 minutes at room temperature. Thereaction is stopped by adding two drops of TFA and water. The mixture isapplied to a reverse phase column and analog 223 (FIG. 20IN) is elutedwith acetonitrile: water (1:4).

EXAMPLE 86 Synthesis of Analog 224

Acylfulvene (116 mg) is dissolved in ethanol (4.0 mL) with stiffing,hydroxylamine hydrochloride (84.2 mg) added, Sodium acetate (233 mg)added, then refluxed for 70 minutes at 85° C. The ethanol is removed,then ethyl acetate (10 mL) added to dissolve crude product, then water(10 mL) added, the organic layer is washed with brine, dried overNa₂SO₄, concentrated then chromatographed (20% ethyl acetate: hexane) togive analog 224 (FIG. 20IO) (63.7 mg, 54% yield).

EXAMPLE 87 Synthesis of Analog 225

Illudin S (439 mg) is dissolved in ethanol (15 mL) with stirring,hydroxylamine hydrochloride (233 mg) added, sodium acetate (933 mg)added, then refluxed for 130 minutes at 85° C. The solution is cooled toroom temperature, filtered, ethanol is removed, then ethyl acetate (30mL) added to dissolve crude product, then water (30 mL) added, theorganic layer is washed with brine, dried over Na₂SO₄, concentrated thenchromatographed (30%->50%, acetone: hexane) to give analog 225 (FIG.20IP) (372 mg, 80% yield).

EXAMPLE 88 Synthesis of Analog 226

Irofulven (37.6 mg) is dissolved with stiffing in CH₂Cl₂, elaidic acid(180 mg. 1.3 equivalents) added, DMAP (15 mg) added, cooled to 0° C.,then DCC (180 μL) in CH₂Cl₂ (640 μL) added. Reaction mixture stirred at0° C. for 1 hour, then additional DCC (120 μL) added, and stirred for 2more hours. Mixture chromatographed (20% ethyl acetate: hexane) to giveanalog 226 (FIG. 20IQ) as a yellow oil (50.5 mg, 48% yield).

EXAMPLE 89 Synthesis of Analog 227

Analog 009 (87 mg) is dissolved with stiffing in CH₂Cl₂, elaidic acid(108 mg) added, DMAP (15.4 mg) added, cooled to 0° C., then DCC (0.5 mL)in CH₂Cl₂ (1.5 mL) added. Reaction mixture stirred at 0° C. for 3 hours,then the mixture directly chromatographed (20% ethyl acetate: hexane) togive analog 227 (FIG. 20IR) as a yellow oil (105 mg, 61% yield).

EXAMPLE 90 Synthesis of Analog 228

Illudin S (86 mg) is dissolved with stiffing in CH₂Cl₂, elaidic acid(202 mg) added, DMAP (15.4 mg) added, cooled to 0° C., then DCC (1.0 mL)in CH₂Cl₂ (3.0 mL) added. Reaction mixture stirred at 0° C. for 3 hours,then the mixture directly chromatographed (20% ethyl acetate: hexane) togive analog 228 (FIG. 20IS) as a yellow oil (198 mg, 77% yield).

EXAMPLE 91 Synthesis of Analog 229

The elaidic ester of 0-diphenylphosphine phenol is first prepared bydissolving with stirring in 3.0 mL of CH₂Cl₂ the O-diphenylphosphinephenol (91.3 mg), elaidic acid (94.5 mg, 1 equivalent), DMAP (9.4 mg).The solution is cooled to 0° C. then DDC (0.44 mL, 1.0 M in CH₂Cl₂) isadded with stirring for 3.5 hours. The precipitate is filtered off anddiscarded. The elaidic ester is chromatographed and concentrated todryness then dissolved in THF (1.0 mL). Analog 195 (FIG. 20HL) (26.1 mg)is dissolved in THF (1.0 mL) and water (80 μL) added. The elaidic estersolution is slowly added to the analog 195 (FIG. 20HL) solution withstirring, and reacted for 22 hours at room temperature. The mixture isdirectly chromatographed (30% acetone: hexane) to give analog 229 (FIG.20IT) (22.2 mg, 47% yield).

EXAMPLE 92 Synthesis of Analog 230

Analog 308 (FIG. 20LU) (22 mg) is dissolved in anhydrous CH₂Cl₂ (1.5mL), diisopropylethylamine (20 μL) added, and the mixture cooled to 0°C., then methylsulfonyl chloride added (15 μL), mixture stirred at 0° C.for 1 hour, and allowed to warm to room temperature while being stirredfor an additional hour. The mixture is chromatographed (30% ethylacetate in hexane) to yield analog 230 (FIG. 20IU) (35% yield).

EXAMPLE 93 Synthesis of Analog 231

Analog 308 (FIG. 20LU) (16 mg) is dissolved in anhydrous CH₂Cl₂ (1.5mL), diisopropylethylamine (20 μL) added, and the mixture cooled to 0°C., then tosyl chloride added (18.4 mg), mixture stirred at 0° C. for 1hour, and allowed to warm to room temperature while being stirred for anadditional 3 hours. The mixture is chromatographed (30% ethyl acetate inhexane) to yield analog 231 (FIG. 20IV) (8.6 mg).

EXAMPLE 94 Synthesis of Analog 240

Analog 232 (FIG. 20IW) (25.1 mg) is dissolved in anhydrous CH₂Cl₂ (2.0mL), 15 μL of acetic anhydride added, and the mixture cooled to roomtemperature, then DMAP added (5 mg), and stirred for 25 minutes. Themixture is partially concentrated then chromatographed (30% ethylacetate in hexane) to yield analog 240 (FIG. 20JE) (26.6 mg, 93% yield).

EXAMPLE 95 Synthesis of Analog 254

Analog 009 (FIG. 20AI) (51.4 mg), 4-carboxybenzene sulfonamide (59.4mg), and DCC (39.6 mg) were dissolved in anhydrous DMF (1.0 mL) at roomtemperature, stirred, then DMAP (15 mg) added. The mixture was stirredfor 2 hours at room temperature then solid material was filtered off.The mixture was then chromatographed (1:1 ethyl acetate: hexane) to giveanalog 254 (FIG. 20JS) (38.6 mg, 45% yield).

EXAMPLE 96 Synthesis of Analog 255

Analog 009 (FIG. 20AI) (244.3 mg) and sulfamoyl chloride (157 mg) weredissolved in anhydrous DMAP (2.0 mL) at room temperature, and stirredfor 3.5 hours. The mixture was concentrated under high vacuum thenchromatographed (30% ethyl acetate in hexane) to give analog 255 (FIG.20JT).

EXAMPLE 97 Synthesis of Analog 259

Analog 255 (FIG. 20JT) (64.7 mg), (diacetoxyiodo)benzene (64.7 mg),dirhodiumtetraacetate or Rh₂(OAc)₄ and magnesium (16.8) dissolved in 5.0mL of CH₂Cl₂ are heated to 70° C. and stirred for 7 hours. The mixtureis filtered, concentrated, then chromatographed (1:1 ethyl acetate:hexane) to give analog 259 (FIG. 20JX).

EXAMPLE 98 Synthesis of Analog 262 and 263 (Prepared Together)

Analog 25 (FIG. 20AY) (44.7 mg) is dissolved in methanol (1.0 mL),Oxone® reagent (246 mg, 3 equivalents) is dissolved in water (1.0 mL).The oxone solution is slowly added to the methanol solution withstirring at room temperature for 3.5 hours, then an additional amount ofOxone reagent added followed by stirring for 1.5 hours. Then 2 mL ofsaturated sodium sulfite solution was added, followed by ethyl acetateextraction, dried over Na₂SO₄, concentrated then chromatographed (1:1Ethyl acetate:hexane) to yield first analog 263 (FIG. 20 KB) (21.4 mg)and then analog 262 (FIG. 20KA) (14.3 mg).

EXAMPLE 99 Synthesis of Analog 284 and 289 (Prepared Together)

Analog 34 (FIG. 20BH) (174 mg) and uracil (227 mg) are dissolved inCH₂Cl₂ with stirring and the mixture cooled to 0° C. Then SnCL₄ (148.8μL) is slowly added. The mixture is stirred at 0° C. for 80 minutes,then concentrated, chromatographed (2->5% methanol: CH₂Cl₂) to giveanalog 284 (FIG. 20KW) (68.9 mg, 33% yield) and analog 289 (FIG. 20LB)(21.6 mg, 10% yield).

EXAMPLE 100 Synthesis of Analog 285

Analog 34 (25 mg) is dissolved in ethanol, andO-(tert-Butyldimethylsilyl) hydroxylamine (25 mg) is added followed bystirring for 2 hours at room temperature. The secondary amineintermediate (9 mg) is recovered by chromatography (30% ethylacetate:hexane), dissolved in CH₂Cl₂, and reacted with sulfamoilchloride (C1SO₂NH₂, 5 mg) and DABCO (2 mg) with stirring for one hour,then additional sulfamoil chloride (6 mg) was added with stirring foranother 1.5 hours. The TPS blocked product was recovered bychromatography (30% ethyl acetate:hexane), and the TPS group was removedin THF by adding TBAF (Tetra-n-butylammonium fluoride). The TPS groupcan also be removed by dissolving the TPS product in pyridine and THF at0° C., then adding HF-pyridine overnight. After TPS deblocking themixture is chromatographed (50% ethyl acetate: hexane) to give analog285 (FIG. 20KX).

EXAMPLE 101 Synthesis of Analog 286 and 287 (Prepared Together)

The ketone groups on 5-fluorouracil are first blocked with TMS groups bydissolving 5-fluorouracil (610 mg) and (NH₄)₂SO₄ in HMDS (10 mL) under anitrogen atmosphere. The solution is refluxed at 142° C. for 2.5 hours,cooled to 60° C. and excess HMDS distilled off, then concentrated todryness under high vacuum. Analog 34 (180 mg) and the di-TMS5-fluorouracil are dissolved in CH₂Cl₂ (5.0 mL) with stiffing and themixture cooled to 0° C. Then SnCL₄ (120 μL) is slowly added drop wise.The mixture is stirred at 0° C. for 3.5 hours, then concentrated,chromatographed (80% ethyl acetate: hexane) to give analog 286 (FIG.20KY) (18.9 mg, 9% yield) and analog 287 (FIG. 20KZ) (84 mg, 38% yield).

EXAMPLE 102 Synthesis of Analog 289

See the preparation of analog 284 for the preparation of analog 289(FIG. 20LB) (284 and 289 prepared simultaneously then separated bychromatography).

EXAMPLE 103

Analogs 299 and 300 (Prepared Together):

Analogs 299(FIG. 20LL) and 300 (FIG. 20LM) are prepared in equal amountsfrom Illudin S using the Mitsunobu reaction. Illudin S is directlyreacted with HN₃ (PPh3, DEAD, benzene) at 0° C. under nitrogen for 45minutes. Mitsunobu, 0. Synthesis 1:1-28, 1981.

EXAMPLE 104 Synthesis of Analog 301

Irofulven (31.6 mg, 0.128 mmol), 5-benzoylvaleric acid (35.8 mg, 0.174mmol) and DMAP (4.7 mg) is dissolved in CH₂Cl₂ (2 mL) under a nitrogenatmosphere, cooled to 0° C., the DCC added (170 μL of 1.0M solution inCH₂Cl₂). The mixture is stirred for 60 minutes then diluted with hexane(10 mL) and filtered. The organic layer is further diluted with CH₂Cl₂,washed with water, then saturated NaHCO₃ then brine, dried with MgSO₄,concentrated, then dissolved in CH₂Cl₂, filtered and chromatographed(10:3 hexane:ethyl acetate), appropriate fractions collected, pooled,concentrated then chromatographed (3:1 hexane:ethyl acetate) to giveanalog 301 (FIG. 20LN) (23.2 mg, 42% yield).

EXAMPLE 105

Analogs 302 and 303 (Prepared Together):

Illudin S (100 mg, 0.378 mmol) is benzoylated by dissolving in pyridine(1.0 mL) then adding 3,5-dintirobenzoyl chloride (110 mg, 0.5 mmol) atroom temperature and stiffing for 24 hours. The mixture is poured ontocrushed ice then extracted with CH₂Cl₂ (10 mL), which is washed twicewith water (20 mL). The organic layer is dried over Na₂SO₄ andconcentrated to yield analogs 302 (FIG. 20LO) and 303 (FIG. 20LP). Thetwo analogs can be separated by column chromatography (1:1 hexane:ethylacetate).

EXAMPLE 106 Synthesis of Analog 304

Analog 009 (84.6 mg) is dissolved in anhydrous CH₂Cl₂ (3.0 mL), DCCadded (81.2 mg), mixture cooled to 0° C., propiolic acid (35 μL) added,then the reaction started with DMAP (15 mg), stirred and allowed to warmto room temperature over 1 hour. The mixture was filtered to removesolids then chromatographed (30% ethyl acetate in hexane) to give analog304 (FIG. 20LQ) (60% yield).

EXAMPLE 107 Synthesis of Analog 305

Analog 009 (99.1 mg) is dissolved in anhydrous CH₂Cl₂ (3.0 mL), pyridine(150 μL) added, then p-nitrophenylchloroformate and stirred for 3.5hours at room temperature. The mixture was concentrated, hexane (20 mL)added, and precipitate filtered before chromatographing (50% ethylacetate in hexane) to give analog 305 (FIG. 20LR) (50% yield).

EXAMPLE 108 Synthesis of Analog 306

Analog 009 (244 mg) is dissolved in anhydrous CH₂Cl₂ (4.0 mL), tosylchloride (181 mg) added, the mixture cooled to 0° C., to which analiquot of pyridine (80 μL) is added. The mixture stirred at 0° C. for 1hour, and allowed to warm to room temperature while being stirred for anadditional 20 hours. The mixture is concentrated then chromatographed(50% ethyl acetate in hexane) to yield analog 306 (FIG. 20LS).

EXAMPLE 109 Synthesis of Analog 307

A solution of 1.0 M N₃H in benzene is first prepared by mixing 654 mgN₃H, 0.65 mL water, in 10 mL of benzene. The mixture is cooled to 0° C.,0.5 mL of concentrated H₂SO₄ added, and allowed to warm slowly to roomtemperature and then stirred for 80 minutes. Next PPh₃ (590 mg) isdissolved in anhydrous THF (1.5 mL) and cooled to 0° C. Then 2.1 mL ofN3H 1.0 M solution is added, followed by DEAD (0.475 mL) then Illudin S(282 mg in 1.0 mL anhydrous THF). The mixture is stirred for 3 hours at0° C., warmed, concentrated, followed by chromatography (30% ethylacetate in hexane) to give analog 307 (FIG. 20LT).

EXAMPLE 110 Synthesis of Analog 308

Analog 307 (FIG. 20LT) (100 mg) is dissolved in anhydrous THF (3.0 mL)at room temperature and PPH3 added (306 mg, 3 equivalents). The mixtureis stirred for 5 hours at room temperature, then the reaction stooped byadding water (0.15 mL). The mixture is heated to 85° C. for 30 minutes,then concentrated and chromatographed (20% methanol in ethyl acetate) togive analog 308 (FIG. 20LU).

EXAMPLE 111 Synthesis of Analog 309

Analog 204 was reacted with HN₃ (DEAD, THF) to yield the azide analog309 (FIG. 20LV) at 68% yield.

EXAMPLE 112 Synthesis of Analog 310

Irofulven (42.9 mg), 4-carboxybenzene sulfonamide (41.4 mg), and DCC(38.4 mg) were dissolved in anhydrous DMF (1.0 mL) at room temperature,stirred and then DMAP (10 mg) added. The mixture was stirred for 75minutes at room temperature then solid material was filtered off. Themixture was then chromatographed (1:1 ethyl acetate: hexane) to giveanalog 310 (FIG. 20LW) (40% yield).

EXAMPLE 113 Synthesis of Analog 311

Illudin M (32.4 mg), 4-carboxybenzene sulfonamide (39.7 mg), and DCC(24.4 mg) were dissolved in anhydrous DMF (1.0 mL) at room temperature,stirred, then DMAP (15 mg) added. The mixture was stirred for 75 minutesat room temperature, allowed to warm to room temperature, then stirredfor 22 hours. The solid material was filtered off and the mixture wasthen chromatographed (1:1 ethyl acetate: hexane) to give analog 311(FIG. 20LX) (35% yield).

EXAMPLE 114 Synthesis of Analog 312

Irofulven (1.18 grams) is dissolved in anhydrous CH₂Cl₂ (4.0 mL), tosylchloride (1.1 equivalent) added, the mixture cooled to 0° C., thenpyridine (0.4 mL) added. The mixture stirred at 0° C. for 1 hour, andallowed to warm to room temperature while being stirred for anadditional 3 hours. The mixture is concentrated then chromatographed(50% ethyl acetate in hexane) to yield analog 312 (FIG. 20LY).

EXAMPLE 115 Synthesis of Analog 313

Analog 308 (FIG. 20LU) (31 mg) is dissolved in anhydrous CH₂Cl₂, cooledto 0° C., with stirring then diisopropylethylamine added (45 μL), thenfluorophenylsulfonyl chloride added (36 μL) for 3 hours at 0° C. Mixtureis directly chromatographed (20% ethyl acetate: hexane) to give analog313 (FIG. 20LZ) (23.3 mg).

EXAMPLE 116 Synthesis of Analog 314

Analog 009 (FIG. 20AI) is dissolved in anhydrous CH₂Cl₂ (4.0 mL), tosylchloride (1.1 equivalent) added, the mixture cooled to 0° C., thenpyridine (0.4 mL) added. The mixture stirred at 0° C. for 1 hour, andallowed to warm to room temperature while being stirred for anadditional 3 hours. The mixture is concentrated then chromatographed(50% ethyl acetate in hexane) to yield analog 314 (FIG. 20MA).

EXAMPLE 117 Synthesis of Analog 315

Irofulven was dissolved in a solution of 2,5 dimethylpyrrole (4 foldexcess molar solution) in 5 mL of dry CH₂Cl₂ at −78° C. Borontrifluoride (equivalent molar amount to the irofulven) was slowly addedwith stirring. The reaction was allowed to stir for 2 more hours at −78°C., then water slowly added. The mixture was extracted twice with 2 foldequivalent volumes of ethyl acetate, the organic extracts combined,washed with saturated NaHCO₃, water, brine, then dried over MgSO₄. Thesolution was concentrated under vacuum until a red residue remained,which was chromatographed on silica gel (50% ethyl acetate in hexane) toyield analog 315 (FIG. 20 MB) (30% yield).

EXAMPLE 118 Synthesis of Analog 316

Analog 316 (FIG. 20MC) was prepared by dissolving Illudin S (20 mg) inpyridine (0.5 mL) and then 4-fluorosulfonylbenzoly chloride (equivalentmolar amount) was added to the mixture in an ice bath. The solution isallowed to warm slowly and then react overnight. The liquid was thenremoved under reduced pressure until a crude residue remained Ratherthan recrystallize from chloroform, the residue was insteadchromatographed on a standard silica gel column usinghexane-ethylacetate (1:1). The mono-adduct (analog 316, FIG. 20MC), adi-adduct and a small amount of unreacted Illudin S were recovered inseparate eluates.

EXAMPLE 119 N₃H 1.0 M Solution

A solution of 1.0 M N3H in benzene is first prepared by mixing 654 mgN₃H, 0.65 mL water, in 10 mL of benzene. The mixture is cooled to 0° C.,0.5 mL of concentrated H₂SO₄ added, and allowed to warm slowly to roomtemperature and then stirred for 80 minutes.

EXAMPLE 120 Synthesis of Analog 193

Irofulven (221 mg, 0.897 umol) is dissolved in anhydrous THF (1.5 mL),then PPh₃ (261 mg, 0.995 umol) is added, then 1.0 M N₃H solution (1.0mL, 1.0 mmol) under nitrogen atmosphere. The solution is cooled to −40°C., and then DIAD (0.21 mL, 1.013 umol) added and stirred for 30 minutesat 0° C. then diluted with hexane, and filtered to remove precipitate.The mixture is concentrated then chromatographed (30% ethyl acetate:hexane) to give analog 193 (FIG. 20HJ) (171 mg, 71%).

EXAMPLE 121 Synthesis of Analog 195

Analog 009 (FIG. 20AI) (31.9 mg, 116 umol) is dissolved in anhydrous THF(3.0 mL), then PPh₃ (33 mg, 126 umol) is added, then 1.0 M N3H solution(0.3061 mL) under nitrogen atmosphere. The solution is cooled to 0° C.,DIAD (30 μL, 145 umol) added and stirred for 30 minutes at 0° C. thenwater (5 μL) is added to destroy the PPh₃. The mixture is concentratedthen chromatographed (30% ethyl acetate: hexane) to give analog 195(FIG. 20HL) (24.9 mg, 72%).

A compound comprising an acylfulvene derivative where R₆ is an amine, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional group, wherethe first functional group is covalently bonded to the amine of R₆ andan AM including one or more primary amino groups, where the AM isselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, where the second functional group is covalently bondedvia an amide bond to at least one of the one or more primary aminogroups of the AM, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an amine, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional group, wherethe first functional group is covalently bonded to the amine of R₆ andan AM including one or more hydroxyl groups, where the AM is selectedfrom the group consisting of an antibody, a peptide, a receptor protein,a growth factor, a lipid, a steroid, folate and an oligonucleotide,where the second functional group is covalently bonded via an esterlinkage to at least one of the one or more hydroxyl groups of the AM, ora pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of a carboxylate group, a carbonyl group, andan aldehyde group, a bi-functional linker with a first terminusincluding a first functional group, and a second terminus including asecond functional group selected from the group consisting of aphotoactivated group, a sulfhydryl group and an amino group, where thefirst functional group is covalently bonded to the R₆ group and an AMincluding one or more primary amino groups, where the AM is selectedfrom the group consisting of an antibody, a peptide, a receptor protein,a growth factor, a lipid, a steroid, folate and an oligonucleotide,where the second functional group is covalently bonded to at least oneof the one or more primary amino groups of the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ includes a fivemembered heterocyclic ring containing both a nitrogen and an oxygen withthe oxygen adjacent to an ether linkage and an AM selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thefive membered heterocylic ring is covalently bonded to the AM throughthe ether linkage, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ includes a fivemembered heterocyclic ring containing both a nitrogen and an oxygen withthe oxygen adjacent to an ether linkage and an AM selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thefive membered heterocylic ring is covalently bonded to the AM throughthe ether linkage, or a pharmaceutically acceptable salt thereof, whereR₆ group is formed from reacting a moiety selected from the groupconsisting of a carboxylate group, a carbonyl group, and an aldehydegroup with a carbodiimide in the presence of an amino acid.

A compound comprising an acylfulvene derivative where R₆ is an aldehydegroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including a second functionalgroup selected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of a carbonyl group, and a ketone group, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional groupselected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an alcoholgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including a second functionalgroup selected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of a sulfhydryl group and a disulfide group, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional groupselected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is a sulfhydrylgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including an amine reactinggroup, where the first functional group is covalently bonded to the R₆group, and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the amine reacting group is covalentlybonded to the AM, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is a sulfhydrylgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including an amine reactinggroup, where the first functional group is covalently bonded to the R₆group, and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the amine reacting group is covalentlybonded to the AM, or a pharmaceutically acceptable salt thereof, whereR₆ group is formed by reducing a disulfide group.

A compound comprising an acylfulvene derivative where R₆ is an alkylhalide and a AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the alkyl halide is displaced in forming acovalent bond with the AM, or a pharmaceutically acceptable saltthereof.

A compound comprising an acylfulvene derivative where R₆ is an alkylhalide and a AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the alkyl halide is displaced in forming acovalent bond with the AM, or a pharmaceutically acceptable saltthereof, where R₆ group is formed by reacting a sulfhydryl group with analkyl halide.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of an acyl azide group, and an azide group andan AM including one or more primary amino groups, where the AM isselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, where the acyl azide group or the azide group iscovalently bonded to at least one of the one or more primary aminogroups of the AM, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an epoxidegroup and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the acylfulvene epoxide derivative iscovalently bonded with the AM through a functional group selected fromthe group consisting of a primary amine, a hydroxyl, and a sulfhydrylgroup, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of an acryloyl group and an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, folate and an oligonucleotide, wherethe acryloyl derivative is covalently bonded with a sulfhydryl group onthe AM forming a stable thioether bond, or a pharmaceutically acceptablesalt thereof.

An AMC comprising a medicant moiety selected from the group consistingof illudin M, and acylfulvene analogs containing a hydroxyl group, alinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, and an oligonucleotide, where thefluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof.

An AMC comprising illudin M containing a hydroxyl group, a linkercovalently bonded at the site of the hydroxyl group through an etherlinkage to form a reactive fluorosulfonyl benzoyl group and an AMselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, where the reactive fluorosulfonyl benzoyl is covalentlybonded with the AM, or a pharmaceutically acceptable salt thereof.

An AMC comprising a toxin containing a hydroxyl group selected from thegroup consisting of ricin A chain, degylcosylated ricin A chain, ricin Bchain, degylcosylated ricin B chain, diphtheria toxin, Pseudomonasaeurginosa exotoxin A, Pseudomonas aeurginosa exotoxin A PE38 fragment,Pseudomonas aeurginosa exotoxin A PE40 fragment, r-gelonin, and saporin,a linker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thereactive fluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof.

An AMC comprising a toxin containing a hydroxyl group selected from thegroup consisting of doxorubicin, auristatin E, auristatin F, monomethylauristatin, maytansine DM1, maytansine DM4, calicheamicin, irinotecan,SN38, pyrrolobenzodiazepines, MGBA, and a duocarmycin derivative, alinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, and an oligonucleotide, where the reactivefluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof.

A compound having the Illudin M 4-FSB structure, or a pharmaceuticallyacceptable salt thereof. A compound having the Illudin M 3-FSBstructure, or a pharmaceutically acceptable salt thereof. A compoundhaving the Illudin M 2-FSB structure, or a pharmaceutically acceptablesalt thereof.

A compound comprising an acylfulvene derivative where R₆ includes a fivemembered heterocyclic ring containing both a nitrogen and an oxygen withthe oxygen adjacent to an ether linkage and an AM selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thefive membered heterocylic ring is covalently bonded to the AM throughthe ether linkage, or a pharmaceutically acceptable salt thereof, wherethe AM is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ includes a fivemembered heterocyclic ring containing both a nitrogen and an oxygen withthe oxygen adjacent to an ether linkage and an AM selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thefive membered heterocylic ring is covalently bonded to the AM throughthe ether linkage, or a pharmaceutically acceptable salt thereof, whereR₆ group is formed from reacting a moiety selected from the groupconsisting of a carboxylate group, a carbonyl group, and an aldehydegroup with a carbodiimide in the presence of an amino acid, where the AMis in the form of a liposomal particle, a nanoparticle, or a PEGylatedcompound, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an aldehydegroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including a second functionalgroup selected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of a carbonyl group, and a ketone group, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional groupselected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an alcoholgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including a second functionalgroup selected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of a sulfhydryl group and a disulfide group, abi-functional linker with a first terminus including a first functionalgroup, and a second terminus including a second functional groupselected from the group consisting of a photoactivated group, asulfhydryl group, and an amino group, where the first functional groupis covalently bonded to the R₆ group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thesecond functional group is covalently bonded to the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is a sulfhydrylgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including an amine reactinggroup, where the first functional group is covalently bonded to the R₆group, and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the amine reacting group is covalentlybonded to the AM, or a pharmaceutically acceptable salt thereof, wherethe AM is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is a sulfhydrylgroup, a bi-functional linker with a first terminus including a firstfunctional group, and a second terminus including an amine reactinggroup, where the first functional group is covalently bonded to the R₆group, and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the amine reacting group is covalentlybonded to the AM, or a pharmaceutically acceptable salt thereof, whereR₆ group is formed by reducing a disulfide group, where the AM is in theform of a liposomal particle, a nanoparticle, or a PEGylated compound,or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an alkylhalide and a AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the alkyl halide is displaced in forming acovalent bond with the AM, or a pharmaceutically acceptable saltthereof, where the AM is in the form of a liposomal particle, ananoparticle, or a PEGylated compound, or a pharmaceutically acceptablesalt thereof.

A compound comprising an acylfulvene derivative where R₆ is an alkylhalide and a AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the alkyl halide is displaced in forming acovalent bond with the AM, or a pharmaceutically acceptable saltthereof, where R₆ group is formed by reacting a sulfhydryl group with analkyl halide, where the AM is in the form of a liposomal particle, ananoparticle, or a PEGylated compound, or a pharmaceutically acceptablesalt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of an acyl azide group, and an azide group andan AM including one or more primary amino groups, where the AM isselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, where the acyl azide group or the azide group iscovalently bonded to at least one of the one or more primary aminogroups of the AM, or a pharmaceutically acceptable salt thereof, wherethe AM is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is an epoxidegroup and an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, folateand an oligonucleotide, where the acylfulvene epoxide derivative iscovalently bonded with the AM through a functional group selected fromthe group consisting of a primary amine, a hydroxyl, and a sulfhydrylgroup, or a pharmaceutically acceptable salt thereof, where the AM is inthe form of a liposomal particle, a nanoparticle, or a PEGylatedcompound, or a pharmaceutically acceptable salt thereof.

A compound comprising an acylfulvene derivative where R₆ is selectedfrom the group consisting of an acryloyl group and an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, folate and an oligonucleotide, wherethe acryloyl derivative is covalently bonded with a sulfhydryl group onthe AM forming a stable thioether bond, or a pharmaceutically acceptablesalt thereof, where the AM is in the form of a liposomal particle, ananoparticle, or a PEGylated compound, or a pharmaceutically acceptablesalt thereof.

An AMC comprising a medicant moiety selected from the group consistingof illudin M, and acylfulvene analogs containing a hydroxyl group, alinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, and an oligonucleotide, where thefluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

An AMC comprising illudin M containing a hydroxyl group, a linkercovalently bonded at the site of the hydroxyl group through an etherlinkage to form a reactive fluorosulfonyl benzoyl group and an AMselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, where the reactive fluorosulfonyl benzoyl is covalentlybonded with the AM, or a pharmaceutically acceptable salt thereof, wherethe AM is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound, or a pharmaceutically acceptable salt thereof.

An AMC comprising a toxin containing a hydroxyl group selected from thegroup consisting of ricin A chain, degylcosylated ricin A chain, ricin Bchain, degylcosylated ricin B chain, diphtheria toxin, Pseudomonasaeurginosa exotoxin A, Pseudomonas aeurginosa exotoxin A PE38 fragment,Pseudomonas aeurginosa exotoxin A PE40 fragment, r-gelonin, and saporin,a linker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thereactive fluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

An AMC comprising a toxin containing a hydroxyl group selected from thegroup consisting of doxorubicin, auristatin E, auristatin F, monomethylauristatin, maytansine DM1, maytansine DM4, calicheamicin, irinotecan,SN38, pyrrolobenzodiazepines, MGBA, and a duocarmycin derivative, alinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl covalently bondedto the hydroxyl group to form an ether linkage and forming a reactivefluorosulfonyl benzoyl group, and an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, and an oligonucleotide, where the reactivefluorosulfonyl benzoyl is covalently bonded with the AM, or apharmaceutically acceptable salt thereof, where the AM is in the form ofa liposomal particle, a nanoparticle, or a PEGylated compound, or apharmaceutically acceptable salt thereof.

A method of generating an AMC comprising the steps of selecting anilludin2 moiety with one or more hydroxyl groups, derivatizing themoiety with a linker selected from the group consisting of4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonylbenzoyl to covalently bond the linker to at least one of the one or morehydroxyl groups forming at least one reactive fluorosulfonyl benzoylgroup, and linking an AM selected from the group consisting of anantibody, a peptide, a receptor protein, a growth factor, a lipid, asteroid, and an oligonucleotide, with the derivatized moiety, where theat least one reactive fluorosulfonyl benzoyl group is covalently bondedwith the AM.

Use of an AMC in the manufacture of a medicant for the treatment ofcancer comprising the steps of selecting an illudin2 moiety with one ormore hydroxyl groups, derivatizing the moiety with a linker selectedfrom the group consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonylbenzoyl and 2-fluorosulfonyl benzoyl to covalently bond the linker to atleast one of the one or more hydroxyl groups forming at least onereactive fluorosulfonyl benzoyl group, and linking an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, and an oligonucleotide, with thederivatized moiety, where the at least one reactive fluorosulfonylbenzoyl group is covalently bonded with the AM.

A method of generating an affinity illudin-S conjugate comprising thesteps of blocking the primary hydroxyl group of illudin S with an acidlabile group that is unreactive with the secondary hydroxyl group ofilludin S, derivatizing the blocked illudin-S with a linker selectedfrom the group consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonylbenzoyl and 2-fluorosulfonyl benzoyl to form a covalent bond with thesecondary hydroxyl group, where the FSB linker forms a reactivefluorosulfonyl benzoyl group, linking an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, with thederivatized the blocked illudin-S where the reactive fluorosulfonylbenzoyl group is covalently bonded with the AM, and deblocking thelinked blocked illudin-S by treating with acid to generate the affinityilludin-S conjugate.

Use of an affinity illudin-S conjugate in the manufacture of a medicantfor the treatment of cancer comprising the steps of blocking the primaryhydroxyl group of illudin S with an acid labile group that is unreactivewith the secondary hydroxyl group of illudin S, derivatizing the blockedilludin-S with a linker selected from the group consisting of4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonylbenzoyl to form a covalent bond with the secondary hydroxyl group, wherethe FSB linker forms a reactive fluorosulfonyl benzoyl group, linking anAM selected from the group consisting of an antibody, a peptide, areceptor protein, a growth factor, a lipid, a steroid, folate and anoligonucleotide, with the derivatized the blocked illudin-S where thereactive fluorosulfonyl benzoyl group is covalently bonded with the AM,and deblocking the linked blocked illudin-S by treating with acid togenerate the affinity illudin-S conjugate.

A method of generating an affinity illudin-M conjugate comprising thesteps of derivatizing illudin-M with a fluorosulfonyl benzoyl halidecompound to generate a reactive fluorosulfonyl benzoyl group, andlinking an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid,folate, and an oligonucleotide, with the derivatized illudin-M where thereactive fluorosulfonyl benzoyl group is covalently bonded with the AM.

Use of an affinity illudin-M conjugate in the manufacture of a medicantfor the treatment of cancer comprising the steps of derivatizingilludin-M with a fluorosulfonyl benzoyl halide compound to generate areactive fluorosulfonyl benzoyl group, and linking an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, folate, and an oligonucleotide, withthe derivatized illudin-M where the reactive fluorosulfonyl benzoylgroup is covalently bonded with the AM.

A compound which induces an activity on a cell population in an animalcomprising an antibody directed to a peptide receptor, where the cellpopulation expresses the peptide receptor, a linker, and an acylfulveneanalog, where the antibody is covalently bound to the acylfulvenethrough the linker, or a pharmaceutically acceptable salt thereof, wherethe compound is cytoactive on the cell population.

A compound which induces an activity on a cell population in an animalcomprising an antibody directed to a peptide receptor, where the cellpopulation expresses the peptide receptor, a linker, and an acylfulveneanalog, where the antibody is covalently bound to the acylfulvenethrough the linker, or a pharmaceutically acceptable salt thereof, wherethe compound is cytoactive on the cell population, where the acylfulveneanalog is selected from the group consisting of analogs 002 (FIG. 20AB),006 (FIG. 20AF), 009 (FIG. 20AI), 015 (FIG. 20AO), 022 (FIG. 20AV), 023(FIG. 20AW), 032 (FIG. 20BF), 056 (FIG. 20CD), 077 (FIG. 20CY), 090(FIG. 20DL), 103 (FIG. 20DY), 106 (FIG. 20EB), 119 (FIG. 20EO), 127(FIG. 20EW), 128 (FIG. 20EX), 145 (FIG. 20FN), 177 (FIG. 20GT), 178(FIG. 20GU), 200 (FIG. 20HQ), 203 (FIG. 20HT), 239 (FIG. 20JD), 244(FIG. 20JI).

A compound which induces an activity on a cell population in an animalcomprising an antibody directed to a peptide receptor, where the cellpopulation expresses the peptide receptor, a linker, and an acylfulveneanalog, where the antibody is covalently bound to the acylfulvenethrough the linker, or a pharmaceutically acceptable salt thereof, wherethe compound is cytoactive on the cell population, where the acylfulveneanalog is toxic to the cell.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅, R₇, R₈ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆ denotes H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉,R₉—OH, —CH₂—N₃, (C₁-C₄)alkyl and R₉ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl, or a pharmaceutically acceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O) CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅, R₇, R₈ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆ denotes H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉,R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉ denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I;R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃₉ CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆denotes H, OH, -, CH₃, CH₂—CH₃₉ CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃₉ CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound of the formula of analog 222.

A compound comprising a toxin selected from the group consisting ofricin A chain, deglycosylated ricin A chain, ricin B chain,deglycosylated ricin B chain, Diphtheria toxin, Pseudomonas aeurginosaexotoxin A, Pseudomonas aeurginosa exotoxin A PE38 fragment, Pseudomonasaeurginosa exotoxin A PE40 fragments, r-gelonin and saporin; a FSBlinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl forming abi-functional reactive fluorosulfonyl benzoyl group forms a covalentbond with a hydroxyl group of the toxin and an AM including one or moreprimary amino groups, where the AM is selected from the group consistingof an antibody, a peptide, a receptor protein, a growth factor, a lipid,a steroid, folate and an oligonucleotide, where the reactivefluorosulfonyl benzoyl group forms an amide bond to at least one of theone or more primary amino groups of the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising a medicant moiety selected from the groupconsisting of doxorubicin, auristatin E, auristatin F, monomethylauristatin, maytansine DM1, maytansineDM4, calicheamicin, irinotecan,SN38, pyrrolobenzodiazepines and MGBA a duocarmycin derivative; a FSBlinker selected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl forming abi-functional reactive fluorosulfonyl benzoyl group forms a covalentbond with a hydroxyl group of the medicant moiety and an AM includingone or more primary amino groups, where the AM is selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thereactive fluorosulfonyl benzoyl group forms an amide bond to at leastone of the one or more primary amino groups of the AM, or apharmaceutically acceptable salt thereof.

An affinity target conjugate comprising a target moiety containing ahydroxyl group; a FSB linker selected from the group consisting of4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonylbenzoyl forming a reactive fluorosulfonyl benzoyl group, where thereactive fluorosulfonyl benzoyl group forms a covalent bond with thehydroxyl group and an AM, where the fluorosulfonyl benzoyl group forms acovalent bond with the AM, or a pharmaceutically acceptable saltthereof.

An affinity target conjugate comprising a target moiety containing ahydroxyl group; a FSB linker selected from the group consisting of4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonylbenzoyl forming a reactive fluorosulfonyl benzoyl group, where thereactive fluorosulfonyl benzoyl group forms a covalent bond with thehydroxyl group and an AM, where the fluorosulfonyl benzoyl group forms acovalent bond with the AM, or a pharmaceutically acceptable saltthereof, where the target moiety is selected from the group consistingof analogs 002 (FIG. 20AB), 006 (FIG. 20AF), 009 (FIG. 20AI), 015 (FIG.20AO), 022 (FIG. 20AV), 023 (FIG. 20AW), 032 (FIG. 20BF), 056 (FIG.20CD), 077 (FIG. 20CY), 090 (FIG. 20DL), 103 (FIG. 20DY), 106 (FIG.20EB), 119 (FIG. 20EO), 127 (FIG. 20EW), 128 (FIG. 20EX), 145 (FIG.20FN), 177 (FIG. 20GT), 178 (FIG. 20GU), 200 (FIG. 20HQ), 203 (FIG.20HT), 239 (FIG. 20JD), 244 (FIG. 20JI).

A compound of formula selected from the group consisting of:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₁, —C(═O)—O—R₁₁,(—O—C═O)—R₁₁, O—R₁₁, CH₂OH, —CH₂OR₁₁, R₁₁—OH, (C₁-C₄)alkyl; R₇, R₈, R₉,R₁₀ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₁₁, (C═O)—O—R₁₁, —O—R₁₁, CH₂OH, —CH₂OR₁₁, R₁₁—OH, (C₁-C₄)alkyl,F, Cl, Br, I; R₄, R₅, R₆ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₁, (C═O)—O—R₁₁, —O—R₁₁, CH₂OH,—CH₂OR₁₁, R₁₁—OH, —CN, (C₁-C₄)alkyl, C₁-C₄—C_(N), and R₁₁ denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S) or nitrogen (N); R₁, R₂, R₃ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₅,—C(═O)—O—R₁₅, (—O—C═O)—R₁₅, O—R₁₅, CH₂OH, —CH₂OR₁₅, R₁₅—OH,(C₁-C₄)alkyl; R₁₀, R₁₁, R₁₂, R₁₃ each independently denote H, OH, -,CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—CH₃)₃, (C(═O) CH₃, (—O—C═O)R₁₅, (C═O)—O—R₁₅, —O—R₁₅, CH₂OH,—CH₂OR₁₅, R₁₅—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₈ denotes H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₅, (C═O)—O—R₁₅, —O—R₁₅, CH₂OH,—CH₂OR₁₅, R₁₅—OH, —CN, (C₁-C₄)alkyl, —C₆(R₁₆)₅, —(C₁-C₄)alkyl-CN,serine, threonine, tyrosine, cysteine; R₄, R₅, R₆, R₇ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₅, (C═O)—O—R₁₅,—O—R₁₅, CH₂OH, —CH₂OR₁₅, R₁₅—OH, —CN, (C₁-C₄)alkyl, —C₆(R₁₆)₅,—(C₁-C₄)alkyl-CN, where R₄, R₅, or R₆ can be absent; R₁₅ denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₆ denotes H, OH,-, CH₃, CH₂—CH₃, where the fluorosulfonyl benzoyl group forms a covalentbond with an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), or sulfur (S); R₁, R₂, R₃ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₈, —C(═O)—O—R₈,(—O—C═O)—R₈, O—R₈, CH₂OH, —CH₂OR₈, R₈—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₈,(C═O)—O—R₈, —O—R₈, CH₂OH, —CH₂OR₈, R₈—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₈denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl, where the fluorosulfonyl benzoyl group forms a covalentbond with an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S) or alkyl nitrogen (i.e., N—R₁₈); Z=oxygen(O) or sulfur (S); X₁=carbon (C), oxygen (O), sulfur (S) or nitrogen(N), where R₇ and R₈ are absent when X₁=oxygen (O) or sulfur (S), whereR₈ is absent when X₁=nitrogen (N); X₂=carbon (C), oxygen (O), sulfur (S)or nitrogen (N), where R₉ and R₁₀ are absent when X₂=oxygen (O) orsulfur (S), where R₁₀ is absent when X₂=nitrogen (N); R₁, R₂, R₃ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₈,—C(═O)—O—R₁₈, (—O—C═O)—R₁₈, O—R₁₈, CH₂OH, —CH₂OR₁₈, R₁₈—OH,(C₁-C₄)alkyl; R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁, R₁₂, R₁₃ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₈,(C═O)—O—R₁₈, —O—R₁₈, CH₂OH, —CH₂OR₁₈, R₁₈—OH, (C₁-C₄)alkyl, where R₄,R₅, R₆, R₁₁, R₁₂, or R₁₃ can be absent; R₁₄, R₁₅, R₁₆, R₁₇ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₈,—C(═O)—O—R₁₈, (—O—C═O)—R₁₈, O—R₁₈, CH₂OH, —CH₂OR₁₈, R₁₈—OH,(C₁-C₄)alkyl, F, Cl, Br, I; R₁₈ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotesCH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); R₁, R₂, R₃, R₇ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₄, —C(═O)—O—R₁₄,(—O—C═O)—R₁₄, O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl; R₄, R₅, R₆,R₇, R₈, R₉, each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₁₄, (C═O)—O—R₁₄, —O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl,—CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, where R₄, R₅, R₆, can be absent; R₁₀,R₁₁, R₁₂, R₁₃ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, —C(═O)R₁₄, —C(═O)—O—R₁₄, (—O—C═O)—R₁₄, O—R₁₄, CH₂OH,—CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, F, Cl,Br, I; R₁₄ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₅ denotes H, OH, -, CH₃,CH₂—CH₃, where the fluorosulfonyl benzoyl group forms a covalent bondwith an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); R₁, R₂, R₃, R₇ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₄, —C(═O)—O—R₁₄,(—O—C═O)—R₁₄, O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl; R₄, R₅, R₆,R₇, each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,(—O—C═O)R₁₄, (C═O)—O—R₁₄, —O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl,—CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, where R₄, R₅, R₆, can be absent; R₈,R₉, R₁₀, R₁₁ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, —C(═O)R₁₄, —C(═O)—O—R₁₄, (—O—C═O)—R₁₄, O—R₁₄, CH₂OH,—CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, F, Cl,Br, I; R₁₄ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₅ denotes H, OH, -, CH₃,CH₂—CH₃, where the fluorosulfonyl benzoyl group forms a covalent bondwith an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S) or alkyl nitrogen (N—R₁₂); Y=nitrogen (N)or oxygen (O), where when Y═O, R₇ is absent; Z=oxygen (O) or sulfur (S);R₁, R₂, R₃ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃,—C(═O)R₁₄, —C(═O)—O—R₁₄, (—O—C═O)—R₁₄, O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH,(C₁-C₄)alkyl; R₄, R₅, R₆, R₇, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₄, (C═O)—O—R₁₄, —O—R₁₄, CH₂OH,—CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, whereR₄, R₅, R₆, can be absent; R₈, R₉, R₁₀, R₁₁, R₁₂ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₄, —C(═O)—O—R₁₄,(—O—C═O)—R₁₄, O—R₁₄, CH₂OH, —CH₂OR₁₄, R₁₄—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₅)₅, F, Cl, Br, I; R₁₄ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₅denotes H, OH, -, CH₃, CH₂—CH₃, where the fluorosulfonyl benzoyl groupforms a covalent bond with an AM, or a pharmaceutically acceptable saltthereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=nitrogen (N) or oxygen (O), where whenX═O, R₇ is absent; R₁, R₂, R₃ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃,—C(═O)CH₃, —C(═O)R₁₂, —C(═O)—O—R₁₂, (—O—C═O)—R₁₂, O—R₁₂, CH₂OH,—CH₂OR₁₂, R₁₂—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇, each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₂, (C═O)—O—R₁₂,—O—R₁₂, CH₂OH, —CH₂OR₁₂, R₁₂—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₃)₅, where R₄, R₅, R₆, can be absent; R₈, R₉, R₁₀, R₁₁ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₂,—C(═O)—O—R₁₂, (—O—C═O)—R₁₂, O—R₁₂, CH₂OH, —CH₂OR₁₂, R₁₂—OH,(C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₃)₅, F, Cl, Br, I; R₁₂denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and R₁₃ denotes H, OH, -, CH₃, CH₂—CH₃, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=nitrogen (N) or oxygen (O), where whenX═O, R₇ is absent; R₁, R₂, R₃ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃,—C(═O)CH₃, —C(═O)R₁₂, —C(═O)—O—R₁₂, (—O—C═O)—R₁₂, O—R₁₂, CH₂OH,—CH₂OR₁₂, R₁₂—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇, each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₂, (C═O)—O—R₁₂,—O—R₁₂, CH₂OH, —CH₂OR₁₂, R₁₂—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₃)₅, where R₄, R₅, R₆, can be absent; R₈, R₉, R₁₀, R₁₁ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₂,—C(═O)—O—R₁₂, (—O—C═O)—R₁₂, O—R₁₂, CH₂OH, —CH₂OR₁₂, R₁₂—OH,(C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₃)₅, F, Cl, Br, I; R₁₂denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and R₁₃ denotes H, OH, -, CH₃, CH₂—CH₃, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=nitrogen (N) or oxygen (O), where whenX═O, R₇ is absent; R₁, R₂, R₃ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃,—C(═O)CH₃, —C(═O)R₁₂, —C(═O)—O—R₁₆, (—O—C═O)—R₁₆, O—R₁₆, CH₂OH,—CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇, R₉, R₁₀ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₆,(C═O)—O—R₁₆, —O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, where R₄, R₅, R₆, can be absent; R₁₂, R₁₃,R₁₄, R₁₅ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃,—C(═O)R₁₆, —C(═O)—O—R₁₆, (—O—C═O)—R₁₆, O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₆—OH,(C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, F, Cl, Br, I; R₁₆denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and R₁₇ denotes H, OH, -, CH₃, CH₂—CH₃, and R₈ denotes acarbon atom, where the fluorosulfonyl benzoyl group forms a covalentbond with an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=nitrogen (N) or oxygen (O), where whenX═O, R₇ is absent; R₁, R₂, R₃ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃,—C(═O)CH₃, —C(═O)R₁₆, —C(═O)—O—R₁₆, (—O—C═O)—R₁₆, O—R₁₆, CH₂OH,—CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇, R₉, R₁₀ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₆,(C═O)—O—R₁₆, —O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, where R₄, R₅, R₆, can be absent; R₁₂, R₁₃,R₁₄, R₁₅ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,—C(═O)R₁₂, —C(═O)—O—R₁₆, (—O—C═O)—R₁₆, O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₂—OH,(C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, F, Cl, Br, I; R₁₆denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and R₁₇ denotes H, OH, -, CH₃, CH₂—CH₃, and R₈ denotes acarbon atom, where the fluorosulfonyl benzoyl group forms a covalentbond with an AM, or a pharmaceutically acceptable salt thereof.

A compound comprising doxorubicin including an amine group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising auristatin including an amine group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising auristatin E including an amine group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=nitrogen (N), sulfur (S) or oxygen(O), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₂,—C(═O)—O—R₁₆, (—O—C═O)—R₁₆, O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₆—OH,(C₁-C₄)alkyl; R₄, R₅, R₆, R₇, R₉, R₁₀ each independently denote H, OH,-, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₆, (C═O)—O—R₁₆, —O—R₁₆, CH₂OH,—CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, whereR₄, R₅, R₆, can be absent; R₁₂, R₁₃, R₁₄, R₁₅ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₆, —C(═O)—O—R₁₆,(—O—C═O)—R₁₆, O—R₁₆, CH₂OH, —CH₂OR₁₆, R₁₆—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₇)₅, F, Cl, Br, I; R₁₆ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₇denotes H, OH, -, CH₃, CH₂—CH₃, and R₈ denotes a carbon atom, and R₁₁denotes a carbon atom, where the fluorosulfonyl benzoyl group forms acovalent bond with an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); R₁, R₂, R₃ each independently denote H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃,CH₂OH, —CH₂OR₁₃, R₁₃—OH, (C₁-C₄)alkyl; R₄, R₅, R₆, R₇, eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃,(C═O)—O—R₁₃, —O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, where R₄, R₅, R₆, can be absent; R₉, R₁₀,R₁₁, R₁₂ each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃,—C(═O)R₁₃, —C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and R₁₄ denotes H, OH, -, CH₃, CH₂—CH₃, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₃,—C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, R₁₃—OH,(C₁-C₄)alkyl; R₆, R₇, R₈, each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₃, (C═O)—O—R₁₃, —O—R₁₃, CH₂OH,—CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, whereR₅, R₆, R₇, can be absent; R₉, R₁₀, R₁₁, R₁₂ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₁₃ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₄denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), where when X═O, or S, then R₈ is absent; R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₁,—(C═O)—O—R₁₁, (—O—C═O)—R₁₁, O—R₁₁, CH₂OH, —CH₂OR₁₁, R₁₁—OH,(C₁-C₄)alkyl; R₆ can denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₁,(C═O)—O—R₁₁, —O—R₁₁, CH₂OH, —CH₂OR₁₁, —R₁₁—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₂)₅, R₇, R₈, R₉, R₁₀ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₁, —C(═O)—O—R₁₁,(—O—C═O)—R₁₁, O—R₁₁, CH₂OH, —CH₂OR₁₁, —R₁₁—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₂)₅, F, Cl, Br, I; R₁₁ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₂denotes H, OH, -, CH₃, CH₂—CH₃ and R₅ denotes a carbon atom, where R₅can be absent, where the fluorosulfonyl benzoyl group forms a covalentbond with an AM, or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₉, —C(═O)—O—R₉,(—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₅, R₆, R₇, R₈each independently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₉,—C(═O)—O—R₉, (—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, —R₉—OH, (C₁-C₄)alkyl,—CN, —(C₁-C₄)alkyl-CN, —C₆(R₁₀)₅, F, Cl, Br, I; R₉ denotes CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl and R₁₀denotes H, OH, -, CH₃, CH₂—CH₃, where the fluorosulfonyl benzoyl groupforms a covalent bond with an AM, or a pharmaceutically acceptable saltthereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); X=oxygen (O), sulfur (S) or Nitrogen(N), when X═O or S, then R₆ is absent; Z=oxygen (O), sulfur (S) orNitrogen (N), when Z═O or S, then R₈ is absent, when Z═N, then R₈═H; n=0to 99; R₁, R₂, R₃ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,—C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃, (—O—C═O)—R₁₃, O—R₁₃, CH₂OH,—CH₂OR₁₃, R₁₃—OH, (C₁-C₄)alkyl; R₉, R₁₀, R₁₁, R₁₂ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅, F, Cl, Br, I; R₄, R₆, R₇ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₃, —C(═O)—O—R₁₃,(—O—C═O)—R₁₃, O—R₁₃, CH₂OH, —CH₂OR₁₃, —R₁₃—OH, (C₁-C₄)alkyl, —CN,—(C₁-C₄)alkyl-CN, —C₆(R₁₄)₅; R₁₃ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotesCH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl; R₁₄ denotes H, OH, -,CH₃, CH₂—CH₃; and R₅ denotes ‘bridging amino acids’ or ‘modifiedbridging amino acids’, where the fluorosulfonyl benzoyl group forms acovalent bond with an AM or a pharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀,(—O—C═O)—R₁₀, O—R₁₀, CH₂OH, —CH₂OR₁₀, R₁₀—OH, (C₁-C₄)alkyl; R₅ candenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₀, (C═O)—O—R₁₀,—O—R₁₀, CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅; R₆, R₇, R₈, R₉ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀, (—O—C═O)—R₁₀, O—R₁₀,CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅, F, Cl, Br, I; R₁₀ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁ 05 and R₁₁ denotes H, OH, -, CH₃, CH₂—CH₃, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM or apharmaceutically acceptable salt thereof.

A compound comprising auristatin F including an amine group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising monomethyl auristatin E including an amine group;a bi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising monomethyl auristatin F including an amine group;a bi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound of formula selected from the group consisting of:

where Y=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀,(—O—C═O)—R₁₀, O—R₁₀, CH₂OH, —CH₂OR₁₀, R₁₀—OH, (C₁-C₄)alkyl; R₅ candenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₁₀, (C═O)—O—R₁₀,—O—R₁₀, CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅; R₆, R₇, R₈, R₉ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀, (—O—C═O)—R₁₀, O—R₁₀,CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅, F, Cl, Br, I; R₁₀ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅ and R₁₁ denotes H, OH, -, CH₃, CH₂—CH₃, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀,(—O—C═O)—R₁₀, O—R₁₀, CH₂OH, —CH₂OR₁₀, R₁₀—OH, (C₁-C₄)alkyl; R₅ candenote —O—, —CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)_CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, —C(═O)R₁₂—, —C(═O)—O—R₁₂—,(—O—C═O)—R₁₀—, O—R₁₂—, —CH₂O—, —CH(OH)—, —O—CH₂—_(—) —CH₂OR₁₂—, —R₁₂—O—,(C₁-C₄)alkyl; R₆, R₇, R₈, R₉ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀, (—O—C═O)—R_(m), O—R₁₀,CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅, F, Cl, Br, I; R₁₀ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅ and R₁₁ denotes H, OH, -, CH₃, CH₂—CH₃; R₁₂ denotes —O—,—CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)—CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, and (C₁-C₄)alkyl, wherethe fluorosulfonyl benzoyl group forms a covalent bond with an AM or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀,(—O—C═O)—R₁₀, O—R₁₀, CH₂OH, —CH₂OR₁₀, R₁₀—OH, (C₁-C₄)alkyl; R₅ candenote —O—, —CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)_CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, —C(═O)R₁₂—, —C(═O)—O—R₁₂—,(—O—C═O)—R₁₀—, O—R₁₂—, —CH₂O—, —CH(OH)—, —O—CH₂—_(—) —CH₂OR₁₂—, —R₁₂—O—,(C₁-C₄)alkyl; R₆, R₇, R₈, R₉ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀, (—O—C═O)—R₁₀, O—R₁₀,CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅, F, Cl, Br, I; R₁₀ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅ and R₁₁ denotes H, OH, -, CH₃, CH₂—CH₃; R₁₂ denotes —O—,—CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)—CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, and (C₁-C₄)alkyl; or apharmaceutically acceptable salt thereof.

A compound comprising maytanasine including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising maytanasine DM1 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising maytanasine DM4 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O), sulfur (S); R₁, R₂, R₃, R₄ each independently denoteH, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₁₀, —C(═O)—O—R₁₀,(—O—C═O)—R₁₀, O—R₁₀, CH₂OH, —CH₂OR₁₀, R₁₀—OH, (C₁-C₄)alkyl; R₅ candenote —O—, —CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)_CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, —C(═O)R₁₂—, —C(═O)—O—R₁₂—,(—O—C═O)—R₁₀—, O—R₁₂—, —CH₂O—, —CH(OH)—, —O—CH₂—_(—) —CH₂OR₁₂—, —R₁₂—O—,(C₁-C₄)alkyl; R₆, R₇, R₈, R₉ each independently denote H, OH, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, —C(═O)R_(m), —C(═O)—O—R₁₀, (—O—C═O)—R₁₀, O—R₁₀,CH₂OH, —CH₂OR₁₀, —R₁₀—OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅, F, Cl, Br, I; R₁₀ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, (C₁-C₄)alkyl, —CN, —(C₁-C₄)alkyl-CN,—C₆(R₁₁)₅ and R₁₁ denotes H, OH, -, CH₃, CH₂—CH₃; R₁₂ denotes —O—,—CH₂—, —CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)—CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, and (C₁-C₄)alkyl; or apharmaceutically acceptable salt thereof.

An Affinity Medicant Conjugate of formula selected from the groupconsisting of:

where R₁, R₂, R₃, R₄ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,—C(═O)CH₃, —C(═O)R₇, —C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇,R₇—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅ denotes a medicant moiety selectedfrom the group consisting of analogs 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,171, 172, 173, 174, 175, 177, 178, 179, 180, 181, 182, 183, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 217, 218,219, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 236, 240,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 262, 263, 267,268, 269, 270, 272, 273, 274, 275, 276, 284, 285, 286, 287, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 and 316; R₆denotes an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, aprotease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the third functional group is covalently bondedto at least one of the one or more primary amino groups of the AM and R₇denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, or a pharmaceuticallyacceptable salt thereof.

A compound comprising calicheamicin including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅, R₇, R₈ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆ denotes H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉,R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃, (C₁-C₄)alkyl and R₉ denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, where thefluorosulfonyl benzoyl group forms a covalent bond with an AM, where theAM is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound, further comprising a physiologically compatibleexcipient, thereby forming a medicant, including racemic mixtures, allenantiomers and mixtures thereof, or a pharmaceutically acceptable saltthereof.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇, R₇—OH, (C₁-C₄)alkyl, F,Cl, Br, I, a heteroatom containing nitrogen (N) or oxygen (O); R₂, R₃,R₄, R₅ each independently denote H, -, CH₃, CH₂OH; and R₆ denotes H, -,CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of the formula:

where R₁ denotes illudin1 or an acylfulvene; R₁ denotes an AM; R₃denotes H, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound comprising irinotecan including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇, R₇—OH, (C₁-C₄)alkyl, F,Cl, Br, I, a heteroatom containing nitrogen (N) or oxygen (O); R₂, R₃,R₄, R₅, R₆ each independently denote H, CH₃, CH₂OH; and R₇ denotes CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₈, (—O—C═O)—R₇, O—R₈, CH₂OH, —CH₂OR₈, R₈—OH, (C₁-C₄)alkyl, F,Cl, Br, I, a heteroatom containing nitrogen (N) or oxygen (O); R₂, R₃,R₄, R₅, R₆ each independently denote H, CH₃, CH₂OH; R₇ denotes an AM;and R₈ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where R₁ denotes either an illudin or an acylfulvene analog selectedfrom the group consisting of analogs 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,171, 172, 173, 174, 175, 177, 178, 179, 180, 181, 182, 183, 188, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 221, 223, 224, 225,226, 227, 228, 229, 230, 231, 236, 240, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 262, 263, 267, 268, 269, 270, 272, 273, 274,275, 276, 284, 285, 286, 287, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314 and 315; and R₂ denotes an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM, where the medicant moiety is selected from the group consistingof illudin M, an illudin1, an illudin2 and an acylfulvene.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM, where the AM includes one or more primary amino groups.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM, where the AM includes one or more thiol groups.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM, where the AM is in the form of a liposomal particle, ananoparticle, or a PEGylated compound.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an AM selected from the group consisting ofan antibody, a peptide, a receptor a protein, a growth factor, a lipid,a steroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the maleimide linker forms a covalent bond withthe AM, where the AMC further comprises a pharmaceutically acceptablesalt.

A compound comprising SN38 including two hydroxyl groups; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of one of the two hydroxyl groups; and an AM,where the fluorosulfonyl benzoyl group forms a covalent bond with theAM, or a pharmaceutically acceptable salt thereof.

A compound of the formula:

where R₁ is selected from a linking unit, or —(CH₂)_(n)—, where n=1 to20, or a substituted alkyl—(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20,where m=0 to 20, and X independently denotes —F, —Cl, —Br, —I, —CN,—CF₃, —SH, —N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H,—NH—C(O)—OH, —C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R₂ denotes an AM selected from the group consisting of anantibody, a peptide, a receptor protein, a growth factor, a lipid, asteroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide; R₃, R₄, R₅, R₆, R₇ each independently denote H, OH,CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃, OC(C═O)CH₂CH₃.

A compound of the formula:

where Z denotes oxygen (O), —NH—, or —CH₂—; R₁ is independently selectedfrom a linking unit, a heteroatom containing nitrogen (N) or oxygen (O),—(CH₂)_(n)—, where n=1 to 20, and a substitutedalkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0 to 20, andX independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH, —N(O), —N(O)₂,—NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH, —C(O)NH₂, —NHS₂H,—S(O)₂NH₂, —OH, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R₂ denotes an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; R₃, R₄, R₅, R₆, R₇ eachindependently denote H, OH, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃,OC(C═O)CH₂CH₃.

A compound of formula selected from the group consisting of:

where R₁ denotes either an illudin or an acylfulvene analog selectedfrom the group consisting of analogs 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,171, 172, 173, 174, 175, 177, 178, 179, 180, 181, 182, 183, 188, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 221, 223, 224, 225,226, 227, 228, 229, 230, 231, 236, 240, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 262, 263, 267, 268, 269, 270, 272, 273, 274,275, 276, 284, 285, 286, 287, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314 and 315; and R₂ denotes an AM selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide.

A compound of formula selected from the group consisting of:

where R₁ is independently selected from a linking unit, —(CH₂)_(n)—, aheteroatom containing nitrogen (N) or oxygen (O), where n=1 to 20, and asubstituted alkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0to 20, and X independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH,—N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH,—C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R₂denotes an AM selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, aprotease cleavable peptide, a glycopeptide, folate and anoligonucleotide; and R₃, R₄, R₅, R₆ each independently denote H, OH,CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃, OC(C═O)CH₂CH₃.

A compound of the formula:

where Z denotes oxygen (O), —NH—, or —CH₂—; R₁ is independently selectedfrom a linking unit, a heteroatom containing nitrogen (N) or oxygen (O),—(CH₂)_(n)—, where n=1 to 20, and a substitutedalkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0 to 20, andX independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH, —N(O), —N(O)₂,—NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH, —C(O)NH₂, —NHS₂H,—S(O)₂NH₂, —OH, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R₂ denotes an AM selected fromthe group consisting of an antibody, a peptide, a receptor protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; R₃, R₄, R₅, R₆, R₇ eachindependently denote H, OH, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃,OC(C═O)CH₂CH₃.

A compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₈—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₈R₉; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈,R₉ each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A compound of the formula:

where X denotes O; R₃, R₅, R₆ and R₇═CH₃; R₄═H; R₁, the LU, is selectedfrom the group consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonylbenzoyl and 2-fluorosulfonyl benzoyl; and R₂ denotes an Affinity Moietyselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, a protease cleavablepeptide, a glycopeptide, folate and an oligonucleotide.

A compound of the formula:

where X denotes O; R₃, R₅ and R₆═CH₃; R₄═H; R₇═CH₂OH; R₁ the LU, isselected from the group consisting of 4-fluorosulfonyl benzoyl,3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl; and R₂ denotes anAffinity Moiety selected from the group consisting of an antibody, apeptide, a receptor protein, a growth factor, a lipid, a steroid, aprotease cleavable peptide, a glycopeptide, folate and anoligonucleotide.

A compound of the formula:

where X denotes a secondary amino group; R₂ denotes an Affinity Moietyselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, a protease cleavablepeptide, a glycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅,R₆, R₇, R₈, R₉ each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl.

A compound of the formula:

where X denotes a carbonyl group; R₂ denotes an Affinity Moiety selectedfrom the group consisting of an antibody, a peptide, a receptor protein,a growth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈,R₉ each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₈—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₈R₉; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the Affinity Moietyincludes one or both one or more primary amino groups and one or morehydroxyl groups; and R₃, R₄, R₅, R₆, R₇, R₈, R₉ each independentlydenote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₈—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₈R₉; R₁denotes a LU selected from the group consisting of 4-fluorosulfonylbenzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl; R₂denotes an Affinity Moiety selected from the group consisting of anantibody, a peptide, a receptor protein, a growth factor, a lipid, asteroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide, where the Affinity Moiety includes one or both one ormore primary amino groups and one or more hydroxyl groups, where theAffinity Moiety is in the form of a liposomal particle, a nanoparticle,or a PEGylated compound and a pharmaceutically acceptable salt thereof;and R₃, R₄, R₅, R₆, R₇, R₈, R₉ each independently denote H, -, CH₃,CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl, further comprising aphysiologically compatible excipient, thereby forming a medicant, andincluding racemic mixtures, all enantiomers and mixtures thereof.

A compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A method of ablating cancer cells comprising the steps of selecting thecompound of compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A method of ablating cancer cells comprising the steps of selecting thecompound of compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl and treating the cancer cells with the compound.

A method of ablating cancer cells comprising the steps of selecting anAM which preferentially binds to a marker present on cancer cells,covalently binding a medicant moiety to the AM via a LU to generate anaffinity medicant conjugate and treating the cancer cells with theaffinity medicant conjugate.

Use of compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₈—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₈R₉; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈,R₉ each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl in the manufacture of a medicament for the treatment ofcancer cells comprising the steps of selecting an AM whichpreferentially binds to a marker present on the cancer cells, covalentlybinding a medicant moiety to the AM via a LU to generate the compoundand treating the cancer cells with the compound.

A compound comprising pyrrolobenzodiazepine including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

Use of compound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a LU; R₂ denotes an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl in the manufacture of a medicament for the treatment ofcancer cells comprising the steps of selecting an AM whichpreferentially binds to a marker present on cancer cells, covalentlybinding a medicant moiety to the AM via a LU to generate the compoundand treating the cancer cells with the compound.

A compound comprising pyrrolobenzodiazepine including an amine group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with the amine group; and an AM, where the fluorosulfonylbenzoyl group forms a covalent bond with the AM, or a pharmaceuticallyacceptable salt thereof.

A compound comprising duocarmycin including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin A including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin B1 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin B2 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin C1 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin C2 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin D including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin SA including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising duocarmycin CC-1065 including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A compound comprising MGBA duocarmycin including a hydroxy group; abi-functional linker selected from the group consisting of 4-FSB,Illudin M, 3-FSB, Illudin M, 2-FSB forming a reactive fluorosulfonylbenzoyl group, where the reactive fluorosulfonyl benzoyl group forms acovalent bond with oxygen of the hydroxy group; and an AM, where thefluorosulfonyl benzoyl group forms a covalent bond with the AM, or apharmaceutically acceptable salt thereof.

A method of ablating cancer cells comprising the steps of selecting acompound of the formula:

where X denotes a heteroatom containing nitrogen (N), oxygen (O) orsulphur (S) including —NH—, —NR₇—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₇R₈; R₁denotes a Linker Unit; R₂ denotes an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide; and R₃, R₄, R₅, R₆, R₇, R₈each independently denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl; and treating the cancer cells with the compound.

A compound comprising an illudin1 selected from the group consisting ofanalogs 189, 190, 217, 218, 219, 222 and 316 including a firstfunctional group and an Affinity Moiety including one or more of one ormore primary amino groups, one or more hydroxyl groups, and one or moresulfhydryl groups, where the Affinity Moiety is selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, folate and an oligonucleotide, where thefirst functional group is covalently bonded to at least one of the oneor more primary amino groups, one or more one or more hydroxyl groups orone or more sulfhydryl groups of the Affinity Moiety, or apharmaceutically acceptable salt thereof.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety, where themedicant moiety is selected from the group consisting of illudin M, anilludin1, an illudin2 and an acylfulvene.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety, where theAffinity Moiety includes one or more primary amino groups.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety, where theAffinity Moiety includes one or more hydroxyl groups.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety, where theAffinity Moiety is in the form of a liposomal particle, a nanoparticle,or a PEGylated compound.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a FSB linker selected from the groupconsisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and2-fluorosulfonyl benzoyl forming a reactive fluorosulfonyl benzoylgroup, where the reactive fluorosulfonyl benzoyl group forms a covalentbond with the hydroxyl group, and an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor a protein, agrowth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the fluorosulfonylbenzoyl group forms a covalent bond with the Affinity Moiety, where theAMC further comprises one or more of a physiologically compatibleexcipient and a pharmaceutically acceptable salt thereof, includingracemic mixtures, all enantiomers and mixtures thereof.

A compound of formula selected from the group consisting of:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O) CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅, R₇, R₈ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆ denotes H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉,R₉—OH, —CH₂—N₃, (C₁-C₄)alkyl and R₉ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where X=oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ each independentlydenote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃,—CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, (C(═O) CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉,CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅, R₇, R₈ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl; R₆ denotes H,OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉,R₉—OH, —CH₂—N₃, (C₁-C₄)alkyl and R₉ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl, further comprising an Affinity Moiety covalent boundwith the fluorosulfonyl benzoyl group, where the Affinity Moiety is inthe form of a liposomal particle, a nanoparticle, or a PEGylatedcompound, a medicant moiety covalently bound with the fluorosulfonylbenzoyl group, a pharmaceutically acceptable salt and one or more of aphysiologically compatible excipient and a pharmaceutically acceptablesalt thereof, including racemic mixtures, all enantiomers and mixturesthereof.

A compound of formula selected from the group consisting of:

where X denotes oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉,—C(═O)—O—R₉, (—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F,Cl, Br, I; R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH,(C₁-C₄)alkyl; R₆ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃,(C₁-C₄)alkyl and R₉ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where X denotes oxygen (O) or sulfur (S); R₁, R₂, R₃, R₄ eachindependently denote H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₉,—C(═O)—O—R₉, (—O—C═O)—R₉, O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, (C₁-C₄)alkyl, F,Cl, Br, I; R₅, R₇, R₈ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,(C(═O)CH₃, (—O—C═O)R₉, (C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH,(C₁-C₄)alkyl; R₆ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, (—O—C═O)R₉,(C═O)—O—R₉, —O—R₉, CH₂OH, —CH₂OR₉, R₉—OH, —CH₂—N₃, —Si(CH₃)₂(CH₃)₃,(C₁-C₄)alkyl and R₉ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl, where the fluorosulfonyl benzoyl group forms a covalentbond with an Affinity Moiety in the form of a liposomal particle, ananoparticle, or a PEGylated compound and the compound further comprisesone or more of a physiologically compatible excipient and apharmaceutically acceptable salt thereof, including racemic mixtures,all enantiomers and mixtures thereof.

A compound of formula selected from the group consisting of:

where R₁ denotes —O—, —CH_(2—), CH₂—CH_(2—), —C—(CH₃)_(2—),—C(CH₃)₂—CH_(2—), —CH₂—CH₂—CH_(2—), —CH₂—CH(CH₃)_CH_(2—), —CH₂—C(CH₃)₂—,—C(═O)CH₂—, —C(═O)R_(8—), —C(═O)—O—R₈—, (—O—C═O)—R₈—, O—R₈—, —CH₂O—,CH(OH)—, —O—CH₂—_(—) —CH₂OR₈—, R₈—O—, (C₁-C₄)alkyl, a heteroatomcontaining nitrogen (N) or oxygen (O); R₂, R₃, R₄, R₅, R₆, R₇ eachindependently denote H, -, CH₃, CH₂OH and R₈ denotes —O—, —CH₂—,—CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH(CH₃)—CH₂—,—CH₂—C(CH₃)₂—, —C(═O)CH₂—, and (C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where R₁ denotes —O—, —CH_(2—), CH₂—CH_(2—), —C—(CH₃)_(2—),—C(CH₃)₂—CH_(2—), —CH₂—CH₂—CH_(2—), —CH₂—CH(CH₃)_CH_(2—), —CH₂—C(CH₃)₂—,—C(═O)CH₂—, —C(═O)R_(8—), —C(═O)—O—R₈, (—O—C═O)—R₈—, O—R₈—, —CH₂O—,CH(OH), —O—CH₂—_(—) —CH₂OR₈—, R₈—O—, (C₁-C₄)alkyl, a heteroatomcontaining nitrogen (N) or oxygen (O); R₂, R₃, R₄, R₅, R₆, R₇ eachindependently denote H, -, CH₃, CH₂OH and R₈ denotes —O—,—CH₂₋₉—CH₂—CH₂—, —C—(CH₃)₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH(CH₃)—CH₂—, —CH₂—C(CH₃)₂—, —C(═O)CH₂—, and (C₁-C₄)alkyl, wherethe azlactone forms a covalent bond with an Affinity Moiety in the formof a liposomal particle, a nanoparticle, or a PEGylated compound and thecompound further comprises one or more of a physiologically compatibleexcipient and a pharmaceutically acceptable salt thereof, includingracemic mixtures, all enantiomers and mixtures thereof.

A compound of formula selected from the group consisting of:

where R₁, R₂, R₃, R₄ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,—C(═O)CH₃, —C(═O)R₇, —C(═O)—O—R₇₉ (—O—C═O)—R₇, O—R₇₉ CH₂OH, —CH₂OR₇,R₇—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅ denotes a medicant moiety; R₆denotes an Affinity Moiety and R₇ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where R₁, R₂, R₃, R₄ each independently denote H, OH, -, CH₃, CH₂—CH₃,CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃,—C(═O)CH₃, —C(═O)R₇, —C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇,R₇—OH, (C₁-C₄)alkyl, F, Cl, Br, I; R₅ denotes a medicant moiety; R₆denotes an Affinity Moiety and R₇ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl, where the Affinity Moiety is selected from the groupconsisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where R₆ is one of the oneor more primary amino groups of the Affinity Moiety, the medicant moietyis selected from the group consisting of analogs 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 171, 172, 173, 174, 175, 177, 178, 179, 180, 181, 182,183, 188, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 221,223, 224, 225, 226, 227, 228, 229, 230, 231, 236, 240, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 262, 263, 267, 268, 269, 270,272, 273, 274, 275, 276, 284, 285, 286, 287, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314 and 315, the Affinity Moiety is in theform of a liposomal particle, a nanoparticle, or a PEGylated compound,and the affinity medicant conjugate further comprises one or more of aphysiologically compatible excipient and a pharmaceutically acceptablesalt thereof, including racemic mixtures, all enantiomers and mixturesthereof.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇, R₇—OH, (C₁-C₄)alkyl, F,Cl, Br, I, or a heteroatom containing nitrogen (N) or oxygen (O); R₂,R₃, R₄, R₅ each independently denote H, -, CH₃, CH₂OH; and R₆ denotes H,-, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₇, (—O—C═O)—R₇, O—R₇, CH₂OH, —CH₂OR₇, R₇—OH, (C₁-C₄)alkyl, F,Cl, Br, I, or a heteroatom containing nitrogen (N) or oxygen (O); R₂,R₃, R₄, R₅, R₆ each independently denote H, -, CH₃, CH₂OH; and R₇denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂,—CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and (C₁-C₄)alkyl.

A compound of the formula:

where R₁ denotes H, OH, -, CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —C(═O)R₇,—C(═O)—O—R₈, (—O—C═O)—R₇, O—R₈, CH₂OH, —CH₂OR₈, R₈—OH, (C₁-C₄)alkyl, F,Cl, Br, I, or a heteroatom containing nitrogen (N) or oxygen (O); R₂,R₃, R₄, R₅, R₆ each independently denote H, -, CH₃, CH₂OH; R₇ denotes anAffinity Moiety; and R₈ denotes CH₃, CH₂—CH₃, CH—(CH₃)₂, —C(CH₃)₃,—CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH, and(C₁-C₄)alkyl.

A compound of formula selected from the group consisting of:

where R₁ is selected from a linking unit, a heteroatom containingnitrogen (N), oxygen (O) or Sulphur (S), or —(CH₂)_(n)—, where n=1 to20, or a substituted alkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20,where m=0 to 20, and X independently denotes —F, —Cl, —Br, —I, —CN,—CF₃, —SH, —N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H,—NH—C(O)—OH, —C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R₂, R₃, R₄, R₅ denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (O—C₄)alkyl and R₆ denotes an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide.

A compound of formula selected from the group consisting of:

where R₁ is selected from a linking unit, a heteroatom containingnitrogen (N), oxygen (O) or Sulphur (S), or —(CH₂)_(n)—, where n=1 to20, or a substituted alkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20,where m=0 to 20, and X independently denotes —F, —Cl, —Br, —I, —CN,—CF₃, —SH, —N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H,—NH—C(O)—OH, —C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R₂, R₃, R₄, R₅, R₆ denote H, -, CH₃, CH₂—CH₃, CH—(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, (C(═O)CH₃, CH₂OH,and (C₁-C₄)alkyl and R₇ denotes an Affinity Moiety selected from thegroup consisting of an antibody, a peptide, a receptor protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide.

An affinity medicant conjugate comprising a medicant moiety containing ahydroxyl group, a maleimide linker forming a covalent bond with thehydroxyl group and an Affinity Moiety selected from the group consistingof an antibody, a peptide, a receptor a protein, a growth factor, alipid, a steroid, a protease cleavable peptide, a glycopeptide, folateand an oligonucleotide, where the maleimide linker forms a covalent bondwith the Affinity Moiety.

An affinity medicant conjugate comprising a medicant moiety containing ahydroxyl group, a maleimide linker forming a covalent bond with thehydroxyl group and an Affinity Moiety selected from the group consistingof an antibody, a peptide, a receptor a protein, a growth factor, alipid, a steroid, a protease cleavable peptide, a glycopeptide, folateand an oligonucleotide, where the maleimide linker forms a covalent bondwith the Affinity Moiety, where the medicant moiety is selected from thegroup consisting of illudin M, an illudin1, an illudin2 and anacylfulvene.

An affinity medicant conjugate comprising a medicant moiety containing ahydroxyl group, a maleimide linker forming a covalent bond with thehydroxyl group and an Affinity Moiety selected from the group consistingof an antibody, a peptide, a receptor a protein, a growth factor, alipid, a steroid, a protease cleavable peptide, a glycopeptide, folateand an oligonucleotide, where the maleimide linker forms a covalent bondwith the Affinity Moiety, where the Affinity Moiety includes one or moreprimary amino groups.

An affinity medicant conjugate comprising a medicant moiety containing ahydroxyl group, a maleimide linker forming a covalent bond with thehydroxyl group and an Affinity Moiety selected from the group consistingof an antibody, a peptide, a receptor a protein, a growth factor, alipid, a steroid, a protease cleavable peptide, a glycopeptide, folateand an oligonucleotide, where the maleimide linker forms a covalent bondwith the Affinity Moiety, where the Affinity Moiety includes one or morethiol groups.

An affinity medicant conjugate (AMC) comprising a medicant moietycontaining a hydroxyl group, a maleimide linker forming a covalent bondwith the hydroxyl group and an Affinity Moiety selected from the groupconsisting of an antibody, a peptide, a receptor a protein, a growthfactor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide, where the maleimide linkerforms a covalent bond with the Affinity Moiety, where the AffinityMoiety is in the form of a liposomal particle, a nanoparticle, or aPEGylated compound and the AMC further comprises a pharmaceuticallyacceptable salt.

A compound of the formula:

where R₁ is selected from a linking unit, a heteroatom containingnitrogen (N) or oxygen (O), or —(CH₂)_(n)—, where n=1 to 20, or asubstituted alkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0to 20, and X independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH,—N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH,—C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R₂denotes an Affinity Moiety selected from the group consisting of anantibody, a peptide, a receptor protein, a growth factor, a lipid, asteroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide; R₃, R₄, R₅, R₆, R₇ each independently denote H, OH, -,CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃, OC(C═O)CH₂CH₃.

A compound of the formula:

where Z denotes oxygen (O), —NH—, or —CH₂—; R₁ is independently selectedfrom a linking unit, a heteroatom containing nitrogen (N) or oxygen (O),—(CH₂)_(n)—, where n=1 to 20, and a substitutedalkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0 to 20, andX independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH, —N(O), —N(O)₂,—NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH, —C(O)NH₂, —NHS₂H,—S(O)₂NH₂, —OH, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R₂ denotes an Affinity Moietyselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, a protease cleavablepeptide, a glycopeptide, folate and an oligonucleotide; R₃, R₄, R₅, R₆,R₇ each independently denote H, OH, -, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃,OC(C═O)CH₃, OC(C═O)CH₂CH₃.

A compound of formula selected from the group consisting of:

where R₁ denotes either an illudin or an acylfulvene analog selectedfrom the group consisting of analogs 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,171, 172, 173, 174, 175, 177, 178, 179, 180, 181, 182, 183, 188, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 212, 213, 214, 215, 216, 221, 223, 224, 225,226, 227, 228, 229, 230, 231, 236, 240, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 262, 263, 267, 268, 269, 270, 272, 273, 274,275, 276, 284, 285, 286, 287, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314 and 315; and R₂ denotes an Affinity Moiety selectedfrom the group consisting of an antibody, a peptide, a receptor protein,a growth factor, a lipid, a steroid, a protease cleavable peptide, aglycopeptide, folate and an oligonucleotide.

A compound of the formula:

where R₁ is independently selected from a linking unit, a heteroatomcontaining nitrogen (N) or oxygen (O), —(CH₂)_(n)—, where n=1 to 20, anda substituted alkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, wherem=0 to 20, and X independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH,—N(O), —N(O)₂, —NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH,—C(O)NH₂, —NHS₂H, —S(O)₂NH₂, —OH, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R₂denotes an Affinity Moiety selected from the group consisting of anantibody, a peptide, a receptor protein, a growth factor, a lipid, asteroid, a protease cleavable peptide, a glycopeptide, folate and anoligonucleotide; and R₃, R₄, R₅, R₆ each independently denote H, OH, -,CH₃, CH₂CH₃, OCH₃, OCH₂CH₃, OC(C═O)CH₃, OC(C═O)CH₂CH₃.

A compound of the formula:

where Z denotes oxygen (O), —NH—, or —CH₂—; R₁ is independently selectedfrom a linking unit, a heteroatom containing nitrogen (N) or oxygen (O),—(CH₂)_(n)—, where n=1 to 20, and a substitutedalkyl-(CH₂)_(n)—C(X)H—(CH₂)_(m)—, where n=0 to 20, where m=0 to 20, andX independently denotes —F, —Cl, —Br, —I, —CN, —CF₃, —SH, —N(O), —N(O)₂,—NH₂, —C(O)H, —C(O)OH, —N═NH, —NH—C(O)H, —NH—C(O)—OH, —C(O)NH₂, —NHS₂H,—S(O)₂NH₂, —OH, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R₂ denotes an Affinity Moietyselected from the group consisting of an antibody, a peptide, a receptorprotein, a growth factor, a lipid, a steroid, a protease cleavablepeptide, a glycopeptide, folate and an oligonucleotide; R₃, R₄, R₅, R₆,R₇ each independently denote H, OH, -, CH₃, CH₂CH₃, OCH₃, OCH₂CH₃,OC(C═O)CH₃, OC(C═O)CH₂CH₃.

The foregoing description of embodiments of the methods, systems, andcomponents of the present invention has been provided for the purposesof illustration and description. It is not intended to be exhaustive orto limit the invention to the precise forms disclosed. Manymodifications and variations will be apparent to one of ordinary skillin the relevant arts. For example, steps performed in the embodiments ofthe invention disclosed can be performed in alternate orders, certainsteps can be omitted, and additional steps can be added. The embodimentswere chosen and described in order to best explain the principles of theinvention and its practical application, thereby enabling others skilledin the art to understand the invention for various embodiments and withvarious modifications that are suited to the particular usedcontemplated. Other embodiments are possible and are covered by theinvention. Such embodiments will be apparent to persons skilled in therelevant art(s) based on the teachings contained herein. The breadth andscope of the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

Table IA shows acylfulvene amine analogs which can be attached to abi-functional linker which can then be attached to a sulfhydryl reactinggroup of the AM using the reagent.

Amine analog Reagent 97 2IT [2-iminothiolane (generated from) methyl4-mercaptobutyrimidate] 121 AMAS [N-(α-maleimidoacetoxy)-succinimideester] 176 BMPA [N-β-malemidopropionic acid] 179 BMPS[N-β-malemidopropyloxy)succinimide ester] 184 C6-SFB [C6-succinimidyl4-formylbenzoate] 203 Citiolone [N-acetylhomocysteinethiolactone] 205DST [disuccinimidyl tartrate] 206 EMCH [N-(episilon-maleimidocaproicacid) hydrazide] 207 EMCS [N-(episilon-maleimideocaproyloxy)succinimideester] 211 GMBS [N-(gamma-maleimideobutyrloxy)succinimide ester] 220KMUA [N-kappa-maleimidoundecanoic acid] 244 KMUH[N-(kappa-maleimidoundecanoic acid) hydrazide] 245 LC-SMCC [succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6- 254 amidocaproate)] 255LC-SDPD [succinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate]264 MBS [m-maleimidobenzoyl-N-hydroxysuccinimide ester] 266 MCP [methyl3-mercaptopropionimidate] 267 MPBH [4-(4-N-maleimidophenyl)-butyric acidhydrazide] 270 M2C2H[4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide] 276 NPIA[p-nitrophenyl iodoacetate] 283 PDPH [3-(2-pyridyldithio)propionylhydrazide] 285 PDTP [3-2(pyridyldithio)propionate] 294 PMPI[N-(p-maleimidophenyl)isocyanate] 295 SATA [succinimidyllS-acetylthioacetate] 296 SATP [succinimidyl acetylthiopropionate] 297SFB [succinimidyl p-formylbenzoate] 308 SFPA [succinimidylp-formylphneoxyacetate] 310 SHTH [succinimidyl 4-hydrazidoterephthalate]311 SIAB [N-succinimidyl(4-iodoacetyl)-aminobenzoate] SIAC [succinimidyl4-(((iodoacetyl)amino)methyl)-cyclohexane-1-caroxylate] SIACX[succinimidyl 6-((((4(iodoacetyl)amino)methyl) cyclohexane-1-carbonyl)aminohexanoate] SIAX [succinimidyl6-((iodoacetyl)amino)hexanoate] SIAXX [succinimidyl6-(6-(((iodoacetyl)amino)-hexanoyl)aminohexanoate] SAMSA[S-acetylmercaptosuccinic anhydride] SMCC [succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate] SM(PEG)2[NHS-PEO₂-maleimide or maleimide PEG2 N-hydroxysuccinimide] SM(PEG)4[NHS-PEO₄-maleimide or maleimide PEG4 N-hydroxysuccinimide] SM(PEG)8[NHS-PEO₈-maleimide or maleimide PEG6 N-hydroxysuccinimide] SM(PEG)12[NHS-PEO₁₂-mleimide or maleimide PEG8 N-hydroxysuccinimide] SMPB[succinimidyl 4-(para-maleimido-phenyl)butyrate] SMPH[succinimidyl-6-(beta-maleimidopropionamido)hexanoate] SMPT[4-succinimidyloxycarbonyl-methyl-alpha-(2-pyridyldithio)toluene] SPDP[N-succinimidyl 3-(2-pyridyldithio)propionate] Sulfo-DST[sulfo-disuccinimidyl tartrate] Sulfo-EMCS[N-(episilon-maleimidocaproyloxy)sulfosuccinimide] Sulfo-GMBS[N-(gamma-maleimidobutyrloxy)sulfosuccinimide ester] Sulfo-KMUS[N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester] Sulfo-LC-SMPT[sulfosuccinimidyl 6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate] Sulfo-LC-SPDP [sulfosuccinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate] Sulfo-MBS[m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester] Sulfo-SIAB[sulfo-succinimidyl(4-iodoacetyl)-aminobenzoate Sulfo-SMCC[sulfosuccimidyl 4-(N-maleimido-methyl)cyclohexane-1- carboxylate]Sulfo-SMPB [sulfosuccimidyl 4-(p-maleimidophenyl)butyrate]

TABLE IB Acylfulvene amine analogs attached to a linker which isattached to a photoactivatable group at the other terminus. Amine analogReagent 97 ANB-NOS [N-5-azido-2-nitrobenzyloxy-succinimide] 121 NHS-ASA[N-hydroxysuccinimidyl-4-azidosalicylic acid] 176 SADPH [N-succinimidyl(4′-azidophenyl)1,3′-dithiopropionate] 179 SANPAH [N-succinimidyl6-(4′azido-2′-nitrophenylamino)hexanoate] 184 SPB[succinimidyl-(4-psoralen-8y;oxy)butyrate] 203 Sulfo-HSAB[N-hydroxysulfosuccinimidyl-4-azidobenzoate] 205 Sulfo-NHS-LC-ASA[sulfosuccinimidyl (4-azido-salicylamido)hexanoate] 206 Sulfo-SADP[sulfosuccinimidyl(4-azido-phenyldithio)propionate] 207 Sulfo-SAED[sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3- 211acetamido)ethyl-1,3′-dithiopropionate] 220 Sulfo-SASD [sulfosuccinimidyl2-(p-azido-salicylamido)ethyl 1,3′- 244 dithiopropionate] 245 Sulfo-SFAD[sulfosuccinimidyl (perfluoroazidobenzamido)ethyl 1,3′- 254dithiopropionate] 255 Sulfo-SAND [sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)ethyl 1,3′- 264 dithiopropionate] 266Sulfo-SANPAH [sulfosuccinimidyl 6-(4′-azido-2′- 267nitrophenylamino)hexanoate] 270 276 283 285 294 295 296 297 308 310 311

TABLE IC Acylfulvene amine analogs attached to a linker which isattached to an amine reactive group at the other terminus. Amine analogReagent 97 BS2G-do [bis(sulfosuccinimidyl)glutarate-d0] 121 BS2G-d4[bis(sulfosuccinimidyl)2,2,4,4,glutarate-d4] 176 BS3 (or Sulfo-DSS)[bis(sulfosuccinimidyl)suberate] 179 BS3do[bis(sulfosuccinimidyl)suberate] 184 BS3d4[bis(sulfosuccinimidyl)2,2,7,7-suberate-d4] 203 BS(PEG)5 [bis(NHS)PEO5]205 BSOCOES [bis(2-(succininidoxycarbonyloxy)ethyl)sulfone] 206 DMA[dimethyl adipimidate] 207 DMP [dimethyl pimelimidate] 211 DMS [dimethylsuberimidate] 220 DFDNB [1,5,-difluoro-2,4-dinitrobenzene] 244 DFDNPS[4,4′-difluoro-3,3′-dinitrophenylsulfone] 245 DSG [disuccinimiylglutarate]; 254 DSS [disuccinimiyl suberate]; 255 DST [disuccinimiyltartarate] 264 DSP or Lomant's reagent [dithiobis(succimidylpropionate)]266 DTBP [dimethyl 3,3′-dithiobispropionimidate] 267 DTSSP (sulfo-DSP) =[3,3'-dithio- 270 bis(sulfosuccinimidylpropionate)] 276 EGS [ethyleneglycol bis(succinimidylsuccinate)] 283 PMPI[N-(4-isocyanatophenyl)maleimide] 285 Sulfo-EGS {ethylene glycolbis(sulfo-succinimidylsuccinate)] 294 295 296 297 308 310 311

TABLE ID Acylfulvene amine analogs attached to a linker which isattached to a reactive group capable of reacting with an aldehyde,carbonyl or carboxylate group at the other terminus. Amine analogReagent 97 C6-SANH [C6-succinimidyl 4-hydraznonicotinate acetonehydrazone] 121 SAN H [succinimidyl 4-hydraznonicotinate acetonehydrazone] 176 EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride] 179 184 203 205 206 207 211 220 244 245 254 255 264 266267 270 276 283 285 294 295 296 297 308 310 311

Table IIA shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM.

Carboxylate analog Reagent 29 37 BMPH [N-β-maleimidopropionic acid)hydrazide-trifluoroacetic acid salt] 38 EMCH[N-(episilon-maleimidocaproic acid) hydrazide] 64 KMUH[N-(kappa-maleimidoundecanoic acid)hydrazide] 97 MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide] 98 PDPH[3-(2-pyridyldithio)propionylhydrazide] 106 SHTH [succinimidyl4-hydrazidoterephthalate] 117 M2C2H [4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide] 118 PMPI[N-(4-Isocyanatophenyl)maleimide] 145 AMBH[2-acetamido-4-mercaptobutyric acid hydrazide] 160 162 177 178 181 258

Table IIB shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM.

Carboxylate analog Reagent 29 37 ABH [p-azidobenzoyl hydrazide] 38 ASBA[4-(p-azidosalicylamido)-butylamine] 64 97 98 106 117 118 145 160 162177 178 181 258

Table IIC shows acylfulvene carboxylate analogs which can be attached toa bi-functional linker, where the linker also contains an amino reactivegroup which can attach to the AM.

Carboxylate analog Reagent 29 37 EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 38 CMC[1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide] 64 AADH [adipic aciddihydrazide] 97 Woodward's Reagent K[N-ethyl-3-phenylisoxazolium-3′sulfonate] 98 106 117 118 145 160 162 177178 181 258

TABLE IID Acylfulvene carboxylate analog attached through carboxylategroup to a linker where the linker also contains an azlactone reactivegroup to attach to the AM. Carboxylate analog Reagent 29 glycine oreither an L or D amino acid including alanine, serine, threonine, 37cysteine, valine, leucine, isoleucine, methionine, proline,phenylalanine, 38 tyrosine, tryptophan, aspartic acid, glutamic acid,apsparagine, glutamine, 64 histidine, lysine, arginine, or nonstandardamino acids including homocysteine, 97 selenocysteine, pyrrolysine,carnitine, hypusine, lanthionine, 2-aminoisobutyric 98 acid,dehydroalanine, gamma-aminobutyric acid, ornithine, citrulline, α-Amino-106 n-butyric acid, Norvaline, Norleucine, Pipecolic acid,Alloisoleucine, α,β- 117 diaminopropionic acid, α,γ-diaminobutyric acid,Allothreonine, α-Amino-n- 118 heptanoic acid, Homoserine,β-Amino-n-butyric acid, β-Aminoisobutyric acid, γ- 145 Aminobutyricacid, isovaline, Sarcosine, N-ethyl glycine, N-propyl glycine, N- 160isopropyl glycine, N-methyl alanine, N-ethyl alanine, N-methylβ-alanine, N- 162 ethyl β-alanine, Isoserine, α-hydroxy-γ-aminobutyricacid, diaminopimelic acid, 177 cystathione, aminoisobutyric acid,dehydroalanine, delta-aminolevulinic acid, 4- 178 aminobenzoic acid,Hydroxyproline, Formylmethioinine, lanthionine, djenkolic 181 acid,Pyroglutamic acid, Hypusine, carboxyglutamic acid, penicillamin, 258thialysine, quisqualic acid, canavine, azetidine-2-carboxylic acid, 2-dimethylglycine.

Table IIIA shows acylfulvene carbonyl analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent.

Carbonyl analog Reagent 13 AMBH [2-acetamido-4-mercaptobutyric acidhydrazide 27 BMPH [N-β-maleimidopropionic acid)hydrazide-trifluoroacetic acid salt] 28 EMCH[N-(episilon-maleimidocaproic acid) hydrazide] 51 KMUH[N-(kappa-maleimidoundecanoic acid)hydrazide] 83 MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide] 84 PDPH[3-(2-pyridyldithio)propionylhydrazide] 124 SHTH [succinimidyl4-hydrazidoterephthalate] 131 144 167 184 201 207 232 233 234 235 237238 239 240 243 276 277 278 279 280 281 282 286 287 288 289 294 295 296297 298 301 302 303

Table IIIB shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent.

Carbonyl analog Reagent 13 ABH [p-azidobenzoyl hydrazide] 27 ASBA[4-(p-azidosalicylamido)-butylamine] 28 51 83 84 124 131 144 167 184 201207 232 233 234 235 237 238 239 240 243 276 277 278 279 280 281 282 286287 288 289 294 295 296 297 298 301 302 303

Table IIIC shows acylfulvene carbonyl analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent.

Carbonyl analog Reagent 13 EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 27 CMC[1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide] 28 C6-SANH[C6-succinimidyl 4-hydraznonicotinate acetone hydrazone] 51 SANH[succinimidyl 4-hydraznonicotinate acetone hydrazone] 83 84 124 131 144167 184 201 207 232 233 234 235 237 238 239 240 243 276 277 278 279 280281 282 286 287 288 289 294 295 296 297 298 301 302 303

Table IVA shows acylfulvene aldehyde analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent.

Aldehyde analog Reagent 8 BMPH [N-β-maleimidopropionic acid)hydrazide-trifluoroacetic acid salt] 10 EMCH[N-(episilon-maleimidocaproic acid) hydrazide] 11 KMUH[N-(kappa-maleimidoundecanoic acid)hydrazide] 13 MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide] 41 PDPH[3-(2-pyridyldithio)propionylhydrazide] pg 253 144 SHTH [succinimidyl4-hydrazidoterephthalate] 156 AMBH [2-acetamido-4-mercaptobutyric acidhydrazide] 201 PMPI [N-(4-isocyanataphenyl)maleimide] AMBH[2-acetamido-4-mercaptobutyric acid hydrazide]

Table IVB shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains a photoactivatablereactive group which can attach to the AM using the reagent.

Aldehyde analog Reagent 8 ABH [p-azidobenzoyl hydrazide] 10 ASBA[4-(p-azidosalicylamido)-butylamine] 11 13 41 144 156 201

Table IVC shows acylfulvene aldehyde analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent.

Aldehyde analog Reagent 8 EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] 10 CMC[1-cyclohexyl-3-2(2-morpholinoethyl)carbodiimide] 11 AADH [adipic aciddihydrazide] 13 C6-SANH [C6-succinimidyl 4-hydraznonicotinate acetonehydrazone] 41 SANH [succinimidyl 4-hydraznonicotinate acetone hydrazone]144 Carbohydrazide [1,3-diamonourea] 156 201

Table VA shows acylfulvene alcohol analogs which can be attached to abi-functional linker which can be attached to a sulfhydryl reactinggroup of the AM using the reagent.

Alcohol analog Reagent Illudin S, Illudin M, 2, PMPI[N-(p-maleimidophenyl)isocyanate] 6, 9, 15, 19, 22, 23, 32, 42, 56, 62,63, 77, 78, 81, 90, 99, 103, 117, 118, 119, 127, 128, 135, 136, 145,155, 159, 162, 187, 200, 204, 208, 277 & 279 & 280, 299, 300, 307, 308

Table VB shows acylfulvene alcohol analogs which can be attached to abi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent.

Alcohol analog Reagent Illudin S, Illudin M, 2, CDI[N,N′-carbonyldiimidazole] 6, 9, 15, 19, 22, 23, DSC[N,N′-disuccinimidyl carbonate] 32, 42, 56, 62, 63, 77, HSC[N-hydroxysuccinimidyl chloroformate] 78, 81, 90, 99, 103, 117, 118,119, 127, 128, 135, 136, 145, 155, 159, 162, 187, 200, 204, 208, 277 &279 & 280, 299, 300, 307, 308

Table VIA shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains an amine reactivegroup which can attach to the AM using the reagent.

Sulfhydryl analog Reagent Analog 51 AMAS[N-(α-maleimidoacetoxy)-succinimide ester] Terminal cysteine BMPA[N-β-malemidopropionic acid] or n-acetyl BMPS[N-β-malemidopropyloxy)succinimide ester] cysteine EMCH[N-(episilon-maleimidocaproic acid) hydrazide] EMCS[N-(episilon-maleimideocaproyloxy)succinimide ester] GMBS[N(gamma-maleimideobutyrloxy)succinimide ester] KMUA[N-kappa-maleimidoundecanoic acid] KMUH [N-(kappa-maleimidoundecanoicacid) hydrazide] LC-SMCC [sucinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6- amidocaproate)] LC-SDPD[succinimidyl 6-(3′-(2-pyridyl-dithio)propionamido)hexanoate] MBS[m-maleimidobenzoyl-N-hydroxysuccinimide ester] M2C2H[4-(N-maleimidomethyl)cyclohexanee-1-1carboxyl-hydrazide] MPBH[4-(4-N-maleimidophenyl)-butyric acid hydrazide] PDPH[3-(2-pyridyldithio)propionylhydrazide] PMPI[N-(p-maleimidophenyl)isocyanate] SBAP [succinimidyl3-bromoacetamido)propionate] SHTH [succinimidyl4-hydrazidoterephthalate] SIA [N-succinimidyl iodacetate] SIAB[N-succinimidyl(4-iodacetyl)aminobenzoate] SMCC [succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate] SMPB [succinimidyl4-(para-maleimido-phenyl)butyrate] SMPH[succinimidyl-6-(beta-maleimidopropionamido)hexanoate] SM(PEG)2[NHS—PEO₂-maleimide or maleimide PEG2 N-hydroxysuccinimide] SM(PEG)4[NHS—PEO₄-maleimide or maleimide PEG4 N-hydroxysuccinimide] SM(PEG)8[NHS—PEO₈-maleimide or maleimide PEG6 N-hydroxysuccinimide] SM(PEG)12[NHS—PEO₁₂-mleimide or maleimide PEG8 N-hydroxysuccinimide] SMPH[succinimidyl-6-(beta-maleimidopropionamido)hexanoate] SMPT[4-succinimidyloxycarbonyl-methyl-alpha-(2-pyridyldithio)toluene] SPDP[N-succinimidyl 3-(2-pyridyldithio)propionate] Sulfo-EMCS[N-(episilon-maleimidocaproyloxy)sulfosuccinimide] Sulfo-GMBS[N-(gamma-maleimidobutyrloxy)sulfosuccinimide ester] Sulfo-KMUS[N-(kappa-maleimidoundecanoyloxy)sulfosuccinimide ester] Sulfo-LC-SMPT[sulfosuccinimidyl 6-(alpha-methyl-alpha-(2-[pyridyldithio)-toluamido)hexanoate] Sulfo-LC-SPDP [sulfosuccinimidyl6-(3′-(2-pyridyl-dithio)propionamido)hexanoate] Sulfo-MBS[m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester] Sulfo-SIAB[sulfosuccimidyl(4-iodo-acetyl)aminobenzoate] Sulfo-SMCC[sulfosuccimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate]Sulfo-SMPB [sulfosuccimidyl 4-(p-maleimidophenyl)butyrate]

Table VIB shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a sulfhydrylreacting group which can attach to the AM using the reagent.

Sulfhydryl analog Reagent Analog 51 BMB [1,4-bis-maleimidobutane]Terminal cysteine BMDB [1,4-bis-maleimidyl-2,3-dihydroxybutyrate] orn-acetyl BMH [bis-malimidehexane] cysteine BMOE [bis-maleimideethane]BM[PEO]2 [1,8-bis-malemidodiethyene-glycol] BM[PEO]3[1,11-bis-malemidotriethyene-glycol] Make expanded claims above DPDPB[1,4-di(3′-(2′pyridyldithio)propionamido)butane] DTME[dithio-bis-(sulfosuccinimidylpropionate)] HBVS[1,6-hexane-bis-vinylsulfone]

Table VIC shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains aphotoactivatable reactive group which can attach to the AM using thereagent.

Sulfhydryl analog Reagent Analog 51 APDP[N-(4-(p-azidosalicylamido)butyl)-3′-(2′- Terminal cysteinepyridyldithio)propionamide] or n-acetyl cysteine

Table VID shows acylfulvene sulfhydryl analogs which can be attached toa bi-functional linker, where the linker also contains a carboxylatereactive group which can attach to the AM using the reagent.

Sulfhydryl analog Reagent Analog 51 EMCA [N-(episilon-maleimidocaproicacid)] Terminal cysteine or n-acetyl cysteine

Table VII shows the cytotoxic data IC₅₀ values (micromolar, 2 hourexposure, N=3, mean±SD) for Illudin M, analog 108 and analog 110 forcells expressing the estrogen receptor (ER) (MCF7) and cells notexpressing the ER (HT29).

Analog HT29 (ER Negative) MCF7 (ER positive) Illudin M 0.52 ± 0.10 0.48± 0.13 108 >55 14.1 ± 2.8  110 >19 2.0 ± 0.1

Table VIII shows the activity of PSA cleavable acylfulven analogs (210,215, 216, 221) and precursor analogs against PSA negative and PSApositive cell line (48 hour exposure, N=3; mean±SD; 1050 values in nM).

Prostate Prostate Prostate PC3 DuPro LnCAP Analog (negative PSA) (tracePSA) (positive PSA) Illudin S 16 ± 5 11 ± 3 15 ± 3 204 (Illudin Stosylate) n.t. n.t.  3,300 ± 1,000 207 (9-amine-leucine)  880 ± 330 450± 40 560 ± 60 211 (9-amine) 350 ± 80 280 ± 20 270 ± 50 212 (IlludinM-proline) 120 ± 20 20 ± 2 120 ± 30 213 (Illudin S-tosylate- 2,200 ±100  360 ± 80  900 ± 200 proline) 214 (Illudin S-Pro-Ser- 300 ± 50  90 ±10 190 ± 30 Ser-HOAc) 210 (9-ester linkage/Ac- 4,700 ± 500  3,500 ± 400  810 ± 130 Hyp-Ser-Ser-Chg-Gln- Gln-Ser-Pro) 215 (Illudin S-tosylaten.t. n.t. >20,000 ester/Ac-Hyp-Ser-Ser- Chg-Gln-Gln-Ser-Pro) 216(Illudin M/ester/Ac- 190 ± 10 280 ± 60 190 ± 30 Hyp-Ser-Ser-Chg-Gln-Gln-Ser-Pro) 221 (211/amide or >21,000 13,000 ± 1,000  800 ± 100nonester) Mu-His- Ser-Ser-Lys(Fmoc)- Leu-Gln-Leu n.t. denotes not tested

Table IX showing peptides cleaved by proteases.

Protease Peptide PSA His-Ser-Ser-Lys-Leu-Gln-XMu-His-Ser-Ser-Lys-Leu-Gln-X Mu-His-Ser-Ser-Lys-Leu-Gln-Lys-XMu-His-Ser-Ser-Lys-Leu-EDA-Lys-X Mc-His-Ser-Ser-Lys-Leu-Gln-XMc-His-Ser-Ser-Lys-Leu-Gln-X Hyp-Ala-Ser-Chg-Gln-Ser-XHyp-Ala-Ser-Chg-Gln-Ser-Leu-X Mu-Hyp-Ala-Ser-Chg-Gln-Ser-XMu-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X Mc-Hyp-Ala-Ser-Chg-Gln-Ser-XMc-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-XMu-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-XMc-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-X4-O—Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro-XArg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-X Mu-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-XMc-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-X Mc-Ser-Ser-Lys-Tyr-Gln-Leu-XMu-Ser-Ser-Lys-Tyr-Gln-Leu-X N-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser-LeuMu-N-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser-LeuMc-N-glutaryl-Hyp-Ala-Ser-chGly-Gln-Ser-Leu Caspase-3Asp-Glu-Val-Asp-Pro-X Mu-Asp-Glu-Val-Asp-Pro-X Mc-Asp-Glu-Val-Asp-Pro-XLys-Gly-Ser-Gly-Asp-Val-Glu-Gly-X Mu-Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-XMc-Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-X Cathepsin B PLE-X Gly-Phe-Leu-Gly-XLys-Lys-Phe-D-Ala-X D-Ala-Phe-Lys-Lys-X Mc-Poly-L-glutamic acid-XMc-Gly-Phe-Leu-Gly-X Mc-Lys-Lys-Phe-D-Ala-X Mc-D-Ala-Phe-Lys-Lys-XMu-Poly-L-glutamic acid-X Mu-Gly-Phe-Leu-Gly-X Mu-Lys-Lys-Phe-D-Ala-XMu-D-Ala-Phe-Lys-Lys-X Val-Cit-X FAPLys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-XMu-Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-XMc-Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-X Kallikrein 2Gly-Lys-Ala-Phe-Arg-Arg-X Mu-Gly-Lys-Ala-Phe-Arg-Arg-XMc-Gly-Lys-Ala-Phe-Arg-Arg-X MMP-2/-9/ Glu-Pro-Cit-Gly-Hof-Tyr-Leu-XMu-Glu-Pro-Cit-Gly-Hof-Tyr-Leu-X Mc-Glu-Pro-Cit-Gly-Hof-Tyr-Leu-XGly-Ile-Leu-Gly-Val-Pro-X Mu-Gly-Ile-Leu-Gly-Val-Pro-XMc-Gly-Ile-Leu-Gly-Val-Pro-X Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-XMu-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-XMc-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X MMP-7Lys-Arg-Ala-Leu-Gly-Leu-Pro-Gly Mu-Lys-Arg-Ala-Leu-Gly-Leu-Pro-GlyMc-Lys-Arg-Ala-Leu-Gly-Leu-Pro-Gly Arg-Pro-Leu-Ala-Leu-Trp-Arg-SerMu-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser Mc-Arg-Pro-Leu-Ala-Leu-Trp-Arg-SerTOP Ala-L-L-Ala-L-Ile Mu-Ala-L-L-Ala-L-Ile Mc-Ala-L-L-Ala-L-Ile uPAD-Ala-Phe-Lys or D-Ala-Phe-Lys-PABC Cathepsin KGly-Gly-Pro-Nle-XMu-Gly-Gly-Pro-Nle-X Mc-Gly-Gly-Pro-Nle-X Plasmin D-Ala-Phe-Lys-Lys-XMu-D-Ala-Phe-Lys-Lys-X Mc-D-Ala-Phe-Lys-Lys-X D-Ala-Phe-Lys-XMu-D-Ala-Phe-Lys-X Mc-D-Ala-Phe-Lys-X ThrombinPoly-L-Lys-Gly-D-Phe-Pip-Arg-Ser-Gly-Gly-Gly-Gly- Gly-X TrypsinPoly-L-Lysine-Gly-Ala-Ser-D-Arg-Phe-Thr-Gly-X

In Table IX, the letter ‘X’ denotes the end attached to the medicant,Chg denotes cyclohexyl glycine, Cit denotes citrulline, EDA denotesethanyl-D-Alanine, Hof denotes homophenylalanine, Hyp denotes4-hydroxyproline, Mc denotes morpholinocarbonyl (carboxy-terminalprotecting group), Mu denotes 4-morpholine-carbonyl (amino-terminalprotecting group), Nle denotes norleucine, PABC denotespara-aminobezoylcarboxyl, PLE denotes Poly-L-glutamic acid, Pip denotespiperidine.

Table X shows different Linker Strategies.

Linker Reactive Group* IDer Functional Group Bond product FSB 1220Carboxylate Ester FSB 1220 Hydroxyl Ether Isothiocyanate 1241 PrimaryAmine Isothiourea Isocyanate 1242 Primary amine Isourea Cyanate esterPrimary amine Isourea Acyl Azide 1243 Primary Amine Amide NHS Ester 1244Primary Amine Amide Sulfonyl chloride 1245 Primary Amine SulfonamideTosylate Ester Thiol Thioether Tosylate Ester Primary Amine SecondaryAmine Tosylate Ester Hydroxyl Ether Tresyl Ester Primary AmineSulfonamide Aldehyde Primary Amine Secondary Amine Epoxide Primary AmineSecondary Amine Carboxylate Primary Amine Carbamate Aryl Halide (LikeFluorobenzene) Primary Amine Arylamine Imidoester 1248 Primary AmineAmidine Carbodiimides (eg EDC or CMC) Primary Amine Amide Diimidazoles(like CDI) Primary amine Carbamate Maleic anhydride Primary amine AmideAlkylphosphate Primary Amine Phosphoramidate Succinic anhydride (likeDSC) 1247 Primary Amine Amide Fluorophenyl esters Primary Amine AmideN,N′-disuccinimidyl carbonate Primary Amine CarbamateN-hydroxylsuccinimidyl Primary Amine Carbamate chloroformate Haloalkyl(like Iodoacetyl) Sulfhydryl Thioester Maleimide (like NEM) SulfhydrylThioether MAL I 1210 Sulfhydryl Thioether MAL I 1211 SulfhydrylThioether Maleimide Hexadienes 2 + 4 cycloaddition Aziridine SulfhydrylThioether Acryloyl Sulfhydryl Thioether Flurobenzene Sulfhydryl ArylThioether Pyridyl disulfide Sulfhydryl Disulfide bond5-thio-2-nitrobenzoic acid (TNB) Sulfhydryl Disulfide bond Vinylsulfone(like HBVS) Sulfhydryl Beta-thiosulfonyl Diazoalkane or DiazoacetateCarboxylate Ester N,N′-carbonyl diimidazole Hydroxyl CarbamateIsocyanate Hydroxyl Carbamate Haloacetyl or alkyl halide Hydroxyl EtherAminooxy Aldehyde Oxime Hydroxylamine Aldehyde Oxime Photolysis ArylAzide Nucleophilic addition Photolysis Halogenated Aryl Nucleophilicaddition Azide Azide/copper catalyst Alkene Triazoline Azide/coppercatalyst Alkyne Triazole Aldehyde/NaCNBH3 Primary Amine Secondary AmineAmino acid 1230 Carboxylate/DCC Azlactone Azlactone Primary Amine AmideWoodward's/Carboxylate Primary Amine Amide DSP or DTSSP Primary AminesDisulfide DSS Primary Amines Amide DST and sulfo-DST Primary AminesAmide BSOCOES and sulfo-BSOCOES Primary Amines Amide EGS and sulfo-EGSPrimary Amines Amide DSG Primary Amines Amide DMA Primary AminesAmidines DMP Primary Amines Amidines DMS Primary Amines Amidines DTBPPrimary Amines Disulfide Difluorobenzene derivatives Primary Amines Arylsecondary amines (DFDNB or DFDNPS) Epoxide Sulfhydryl Thioether EpoxideHydroxyl Ether Carbohydrazide Aldehyde Hydrazone-Hydrazine SPDP orSulfo-SPDP or LC- Primary Amine Amide SDPDP or Sulfo-LC-SDPDP SPDP orSulfo-SPDP or LC- Sulfhydryl Disulfide SDPDP or Sulfo-LC-SDPDP SMPT orSulfo-LC-SMPT Primary Amine Amide SMPT or Sulfo-LC-SMPT SulfhydrylDisulfide SMCC or Sulfo-SMCC or LC- Primary Amine Amide SMCC orSulfo-LC-SMCC SMCC or Sulfo-SMCC or LC- Sulfhydryl Disulfide SMCC orSulfo-LC-SMCC MBS and sulfo-MBS Primary Amine Amide MBS and sulfo-MBSSulfhydryl Thioether SIA/B and sulfo-SIA/B Primary Amine Amide SIAB andsulfo-SIAB Sulfhydryl Thioether SIAC or SIACX or SIAX or Primary AmineAmide SIAXX SIAC or SIACX or SIAX or Sulfhydryl Thioether SIAXX GMBS andsulfo-GMBS Primary Amine Amide GMBS and sulfo-GMBS Sulfhydryl ThioetherMPBH Sulfhydryl Thioether MPBH Carbonyl Amide/Hydrazone M2C2H SulfhydrylThioether M2C2H Carbonyl Amide PDPH Sulfhydryl Disulfide PDPH CarbonylAmide/Hydrazone NHS-ASA Primary Amine Photoreactive Aryl AzideSulfo-NHS-ASA Primary Amine Photoreactive Aryl Azide Sulfo-NHS-LC-AS APrimary Amine Photoreactive Aryl Azide HSAB and Sulfo-HSAB Primary AminePhotoreactive Azide with Amide SANPAH and Sulfo-SANPAH Primary AminePhotoreactive Azide with Amide ANB-NOS Primary Amine Photoreactive Azidewith Amide SAND and Sulfo-SAND Primary Amine Photoreactive Azide withAmide SADP and Sulfo-SADP Primary Amine Photoreactive Azide with AmideSAPB and Sulfo-SAPB Primary Amine Photoreactive Azide with Amide SAEDand Sulfo-SAED Primary Amine Photoreactive Azide with Amide Sulfo-SAMCAPrimary Amine Photoreactive Azide with Amide Sulfo-SASD Primary AminePhotoreactive Azide with Amide Sulfo-SFAD Primary Amine PhotoreactiveAzide with Amide pNDPD Primary Amine Photoreactive Azide with AmidePNP-DTP Primary Amine Photoreactive Diazo with Amide APDP SulfhydrylPhotoreactive Azide with Thioether ABH Aldehyde Photoreactive Azide withHydrazone ASBA Carboxylate Photoreactive Azide with Amide SPB PrimaryAmine Photoreactive Psoralen group with Amide PMPA or PMPS SulfyhydrylThioether SANH or SHNH or SHTH Primary Amine Amide SANH or SHNH or SHTHAldehyde Hydrazone BMPA or BMPS Sulfhydryl Thioether BMPA or BMPSPrimary Amine Amide SATA or SATP or SAMSA Primary Amine Amide SATA orSATP or SAMSA Hydroxylamine Sulfhydryl AMBH Aldehyde Hydrazone PMPISulfhydryl Thioether PMPI Hydroyxl Carbamate AADH Aldehyde HydrazoneAMAS Primary Amine Amide AMAS Sulfhydryl Thioether KMUS or Sulfo-KMUSPrimary Amine Amide KMUS or Sulfo-KMUS Sulfhydryl Thioether EMCH or EMCSor sulfo-EMCS Primary Amine Amide EMCH or EMCS or sulfo-EMCS SulfhydrylThioether BS2 or BS3 or BS(PEG)5 series Amine Amide Citiolone PrimaryAmine Amide with free Sulfhydryl SMPB or Sulfo-SMPB or SMPH or PrimaryAmine Amide SBAP SMPB or Sulfo-SMPB or SMPH or Sulfhydryl Thioether SBAPWoodward's Reagent K Carboxylate Enol Ester Intermediate “ “ Enol EsterIntermediate Primary Amine Amide KMUA Sulfhydryl Thioether KMUA PrimaryAmine in Amide presence of EDC KMUH Sulfhydryl Thioether KMUH Aldehydeor Hydrazone Carboxylate BMPH Sulfhydryl Thioether BMPH Aldehyde orHydrazone Carboxylate PDTP Primary Amine Amide SEB or SFPA Primary AmineAmide with free aldehyde SM(PEG)n Series Primary Amine Amide SM(PEG)nSeries Sulfhydryl Thioether DPDPB Two Sulfhydryls Two DisulfidesBM[PEO]n series Two Sulfhydryls Two Thioethers BMH or BMOE TwoSulfhydryls Two Thioethers BMB or BMDB Two Sulfhydryls Two ThioethersDTME Two Sulfhydryls Two Thioethers with internal disulfide bond NPIAPrimary Amine Amide NPIA Sulfhydryl Thioether MCP Primary Amine Amidine• Abbreviation in Table X have been defined previously in Tables Ithrough Table VI.

Table XI shows Illudin1 analogs.

Analog # identified in Entry FIG. 20 1 106 2 107 3 108 4 109 5 110 6 1117 112 8 113 9 114 10 115 11 116 12 117 13 118 14 119 15 120 16 121 17122 18 123 19 124 20 125 21 126 22 127 23 128 24 129 25 130 26 131 27133 28 134 29 135 30 136 31 137 32 138 33 139 34 140 35 141 36 142 37143 38 144 39 145 40 146 41 147 42 148 43 149 44 150 45 151 46 152 47153 48 154 49 155 50 156 51 157 52 158 53 159 54 160 55 161 56 162 57163 58 164 59 165 60 166 61 167 62 168 63 169 64 171 65 172 66 173 67174 68 175 69 177 70 178 71 179 72 180 73 181 74 182 75 183 76 188 77189 78 190 79 191 80 192 81 193 82 194 83 195 84 196 85 197 86 198 87199 88 200 89 201 90 202 91 203 92 204 93 205 94 206 95 207 96 208 97209 98 210 99 212 100 213 101 214 102 215 103 216 104 217 105 218 106219 107 221 108 222 109 223 110 224 111 225 112 226 113 227 114 228 115229 116 230 117 231 118 236 119 240 120 249 121 250 122 251 123 252 124253 125 254 126 255 127 256 128 257 129 258 130 259 131 262 132 263 133267 134 268 135 269 136 270 137 272 138 273 139 274 140 275 141 276 142284 143 285 144 286 145 287 146 289 147 290 148 291 149 292 150 293 151294 152 295 153 296 154 297 155 298 156 299 157 300 158 301 159 302 160303 161 304 162 305 163 306 164 307 165 308 166 309 167 310 168 311 169312 170 313 171 314 172 315 173 316

Table XII shows previously identified Illudin analogs.

Analog # identified in Entry FIG. 20 1 001 2 002 3 003 4 004 5 005 6 0067 007 8 008 9 009 10 010 11 011 12 012 13 013 14 014 15 015 16 016 17017 18 018 19 019 20 020 21 021 22 022 23 023 24 024 25 025 26 026 27027 28 028 29 029 30 030 31 031 32 032 33 033 34 034 35 035 36 036 37037 38 038 39 039 40 040 41 041 42 042 43 043 44 044 45 045 46 046 47047 48 048 49 049 50 050 51 051 52 052 53 053 54 054 55 055 56 056 57057 58 058 59 059 60 060 61 061 62 062 63 063 64 064 65 065 66 066 67067 68 068 69 069 70 070 71 071 72 072 73 073 74 074 75 075 76 076 77077 78 078 79 079 80 080 81 081 82 082 83 083 84 084 85 085 86 086 87087 88 088 89 089 90 090 91 091 92 092 93 093 94 094 95 095 96 096 97097 98 098 99 099 100 100 101 101 102 102 103 103 104 104 105 105 106132 107 170 108 176 109 184 110 185 111 186 112 187 113 211 114 220 115232 116 233 117 234 118 235 119 237 120 238 121 238 122 241 123 242 124243 125 244 126 245 127 246 128 247 129 248 130 260 131 261 132 264 133265 134 266 135 271 136 277 137 278 138 279 139 280 140 281 141 282 142283 143 288

TABLE XIII Summary NCI DTP 60 Cell Line Data. Mean GI50 Mean TGI MeanLD50 NAME/NSC inhibition cytostatic cytotoxic Pyrrolobenzodiazepines 7nM 302 nM >23,000 nM* 694501 Maytansine** 19 nM 318 nM 49,200 nM 153858Fumagillol 6,130 nM 9,850 nM >50,000 nM 642492 Dolstatin-10 17 nM 2,680nM >50,000 nM 376128 Auristatins 1.4 nM 902** nM >5,000 nM** 654663Enadiyne 157365 2,900 nM >100,000 nM >100,000 nM Halichondrin B 1.2 nM199 nM >1,000 nM 609395 Tubulysin A 12 nM 1,318 nM >10,000 nM Illudin S10 nM 64 nM 511 nM Illudin M 3 nM 20 nM 291 nM

TABLE XIV Mechanisms of Drug Resistance. Mechanism of Multi-drugResistance to Illudins, Syn-illudins, and Resistance AcylfulvenesGp170/MDR1 No Gp180/MRP No Topoisomerase I No Topoisomerase II NoMVP/LRP (vault) No Thiol content/GST pi No DNA repair No Myc expressionNo Bcl-2 expression No BRCA status No P53 status No P21 status No MGMTexpression No Microtubulin alteration No

Table XV showing the ability of the Illudin, Syn-Illudin and Acylfulveneanalogs to inhibit tumor cell growth.

Mean IC50 value (nM) ± SD, N = 3 unless otherwise indicated AnalogFigure MV522 Target Cell Line 8392B Nontarget Cell Line Number Label 2hr exposure 48 hr exposure 2 hr exposure 48 hr exposure 001 20AA 2200 ±100 350 ± 20   830 ± 100 002 20AB 110 ± 40 70 ± 10 26000 ± 4500  800 ±100 003 20AC 004 20AD 4200 600 005 20AE 006 20AF 007 20AG 008 20AH 870 ±90 630 ± 80  12200 ± 700  15100 ± 2200 009 20AI 500 ± 30 850 ± 180 47100 ± 11000 43200 ± 2300 010 20AJ  8900 ± 1500 170 ± 60  29400 ± 160014500 ± 1700 011 20AK 4900 ± 900 1200 (N = 2) >100000 40400 ± 6700 01220AL  5150 ± 1350 320 ± 90  42200 ± 5000 18800 ± 2800 013 20AM 5100 ±700 270 ± 130 11900 ± 1300 4200 ± 400 014 20AN 115 ± 30 460 ± 120 9650 ±200 1100 ± 300 015 20AO 1800 ± 200 480 ± 110  810 ± 260 1300 ± 150 01620AP  490 ± 130 440 ± 90  >100000 870 ± 60 017 20AQ 2400 ± 360 320 ± 60 14700 ± 900  018 20AR  8800 ± 2900  4200 ± 1300 019 20AS 470 ± 60 660 ±80  >75000 020 20AT  530 ± 140 230 ± 10  25000 ± 3100 021 20AU  2400 ±1000 930 ± 250 34400 ± 9400 022 20AV  700 ± 200 680 ± 180 31700 ± 1400023 20AW  2900 ± 1140 2750 ± 500  >138000 024 20AX 1800 ± 200 1200 ±300  12800 ± 2100 025 20AY 1300 ± 310 1200 ± 100  >25000 026 20AZ 02720BA 028 20BB 029 20BC 030 20BD >3000 031 20BE >3000 032 20BF  600 ± 190210 ± 30  >30000 033 20BG 10000 ± 1100 4600 ± 200  29900 ± 3300 034 20BH1400 ± 170 490 ± 40  >100000 4400 ± 200 035 20BI 5600 ± 600 >150000 03620BJ 037 20BK 26000 ± 5000 29200 ± 2300  >85000 038 = 20BL 750 ± 6024900 ± 8000 091 039 = 20BM 1500 ± 240 600 ± 40  24600 ± 2400  820 ± 250092 040 = 20BN 3400 ± 360 700 ± 90  24000 ± 3300 5200 ± 470 093 041 20BO042 20BP 043 20BQ 044 20BR 045 20BS 046 20BT 047 20BU 048 20BV 049 20BW050 20BX 051 20BY 052 20BZ 053 20CA 054 20CB 055 20CC 056 20CD 057 20CE058 20CF 059 20CG 060 20CH 19400 ± 1800 27600 ± 3000 061 20CI 062 20CJ2600 ± 300 660 ± 200 37100 ± 2300 063 20CK 43000 ± 5700 580 ± 250 06420CL 28000 ± 4600 1200 ± 300  065 20CM  6200 ± 1100 2500 ± 1200 066 20CN067 20CO 068 20CP 069 20CQ 070 20CR 071 20CS 072 20CT 073 20CU 074 20CV075 20CW 19600 ± 9700 62000 ± 3600 076 20CX 24000 ± 6100 39500 ± 7200077 20CY  9200 ± 1200 078 20CZ 20400 ± 6300 >100000 079 20DA  7700 ±3500 >100000 080 20DB  8800 ± 2400 >100000 081 20DC >80000 >80000 08220DD 50600 ± 7100 >100000 083 20DE 37200 ± 2900  >42000 084 20DF 28200 ±1400  >42000 085 20DG >40000 >40000 086 20DH 087 20DI >40000 24700 ±3900  >40000 088 20DJ 089 20DK 19300 ± 5700 15500 ± 2800  >60000 09020DL 2500 ± 400 2900 ± 400  1600 ± 200 3800 ± 300 091 20DM aka 38 09220DN aka 39 093 20DO aka 40 094 20DP  800 ± 100 210 ± 20   9000 ± 1700110 ± 10 095 20DQ 096 20DR 2700 ± 400 6200 ± 600  >88000 >3000 097 20DS2900 ± 100 >82000 098 20DT 18800 ± 2500 4600 ± 250  >65000 11700 ± 1800099 20DU  8400 ± 1100 1800 ± 200  4000 ± 400 300 ± 20 100 20DV >100001700 ± 500  101 20DW >8000 >7500 102 20DX >13000 1300 ± 100  103 20DY31800 ± 4900 5900 ± 400  12100 ± 2000 2300 ± 200 104 20DZ 6300 ± 4006000 ± 500  36400 ± 6500 2700 ± 600 105 20EA  7300 ± 1200 2100 ±400  >100000 106 20EB  5200 ± 1000 >83000 107 20EC >50000 1600 ±100  >50000 108 20ED 12300 ± 2300 520 ± 50  >55000  6000 ± 1600 10920EE >50000 >50000 110 20EF >55000 1400 ± 100  >55000 25300 ± 2100 11120EG 16700 ± 2100 11900 ± 2800  34600 ± 2100 10200 ± 1000 112 20EH 10000± 2000 6700 ± 1200 14900 ± 100  5200 ± 300 113 20EI 85000 ± 700  14100 ±3000  >93000  7800 ± 1000 114 20EJ 1500 ± 100 260 ± 70  25100 ± 1000 700 ± 100 115 20EK 1500 ± 100 70 ± 5  1600 ± 700 630 ± 60 116 20EL  400± 100 1000 ± 50  7000 ± 400 170 ± 30 117 20EM 1100 ± 100 100 ± 30   7900± 1600 10 ± 2 118 20EN 14000 ± 2000 740 ± 120 24500 ± 4500 2000 ± 400119 20EO 1100 ± 70  270 ± 40  >33000 >10000 120 20EP 2800 ± 900 600 ±100 19100 ± 4600  510 ± 110 121 20EQ 300 ± 10 90 ± 10 15200 ± 6000 1300± 500 122 20ER 6400 ± 300 2400 ± 300  14500 ± 1200 1100 ± 300 123 20ES1900 ± 400 600 ± 60  450 ± 30 2400 ± 500 124 20ET 2800 ± 700 870 ±350 >30000 2400 ± 550 125 20EU 3700 ± 600 1200 ± 200  15500 ± 1400  600± 100 126 20EV 2100 ± 500 900 ± 100 >30000 330 ± 80 127 20EW 870 ± 30340 ± 90  >30000 100 ± 40 128 20EX  840 ± 230 370 ± 50  >35000 800 ± 70129 20EY >136000 19700 ± 1900  >136000 39400 ± 9200 130 20EZ  700 ± 100130 ± 40  27,000 ± 7000  4400 ± 500 131 20FA 132 133 20FB 58800 ± 660015800 ± 2600  12200 ± 2300 2700 ± 400 134 20FC 50000 ± 6000 28000 ±4000  43900 ± 5100  8500 ± 2000 135 20FD 1600 ± 300 22 ± 4   70 ± 20 22± 2 136 20FE 430 ± 10 130 ± 10  >6200 25 ± 2 137 20FF  850 ± 110 1200 ±100   8500 ± 1200 710 ± 60 138 20FG 2100 ± 200 1000 ± 200  5400 ± 200 820 ± 230 139 20FH 6400 ± 900 3400 ± 500  11600 ± 900   2600 ± 1000 14020FI 17100 ± 5100 >14000 12700 ± 300  >14000 141 20FJ 11400 ± 1000 3700± 800  13700 ± 1900 1100 ± 140 142 20FK  90 ± 10 24 ± 7   6400 ± 1100 80± 6 143 20FL  43500 ± 11300 11400 ± 1800   56500 ± 20000 3600 ± 700 14420FM 145 20FN 146 20FO 2500 ± 400 740 ± 280 13,000 ± 1200  14720FP >76000 26100 ± 12900 >76000 43800 ± 3000 148 20FQ 17100 ± 1100 6800± 1100  61000 ± 11600  6700 ± 1600 149 20FR  2900 ± 1000 1500 500 44600± 1400 4100 ± 900 150 20FS  9500 ± 1600 1400 ± 400  59000 ± 5500 10600 ±800  151 20FT 7900 ± 400 4200 ± 1600 25500 ± 1200  6600 ± 2300 152 20FU 6400 ± 1200 49000 ± 7700  9100 ± 100 153 20FV  8700 ± 2700 10900 ±3400  >90000 15800 ± 9600 154 20FW >70000 61300 ± 10000 >70000 46,700 ±13100  155 20FX  8200 ± 1200 3600 ± 400  17,000 ± 4000   9100 ± 1100 15620FY 7200 ± 500 3100 ± 100  32,300 ± 9,400  5500 ± 1200 15720FZ >400,000 >123,000 >350,000 13100 ± 1600 15820GA >175,000 >175,000 >200,000 61,000 ± 9,000 159 20GB 2700 ± 400 120 ±10  13,700 ± 4,200 <10 nM 160 20GC 1900 ± 200 500 ± 200  52,400 ± 17,800 3200 ± 1100 161 20GD 2800 ± 500 3300 ± 700  13,800 ± 3,400 >10,000 16220GE 163 20GF 3500 ± 800 820 ± 40  18600 ± 800   910 ± 100 164 20GG 70 ±10  3500 ± 1600 130 ± 40 165 20GH  7700 ± 1100 290 ± 40  11000 ± 330011000 ± 1000 166 20GI 6500 ± 600 7200 ± 1900  6500 ± 2100  6000 ± 1500167 20GJ 14800 ± 2200 18500 ± 2300 168 20GK 169 20GL 7100 ± 600 2300 ±600 170 20GM 171 20GN 172 20GO 173 20GP 174 20GQ 175 20GR 176 20GS 17720GT 7500 ± 800 1900 ± 800  73000 ± 5000  4100 ± 1300 178 20GU 21000 ±4000 1000 ± 100  32000 ± 9000 >8000 179 20GV 180 20GW 19900 ± 300  >4000 5200 ± 1800 660 ± 50 181 20GX 182 20GY  99000 ± 12000 38000 ± 8200 39000 ± 7000 18700 ± 2700 183 20GZ >120,000 >275,000 >120,000 >235,000184 20HA  800 ± 300 210 ± 20  >100,000 >10000 185 20HB 1700 ± 600 1900 ±100  186 20HC 144000 ± 32000 70000 ± 16000  79000 ± 24000 48000 ± 2000187 20HD 1300 ± 400 900 ± 200 3200 ± 800 3200 ± 700 188 20HE 189 20HF 8900 ± 2500 6100 ± 2600 41,000 ± 3700  190 20HG 19,000 ± 4000  >9,00056,000 ± 2000  >9,000 191 20HH >140,000 49,000 ± 13000 >140,000 15000 ±4000 192 20HI 1,600 ± 200  700 ± 100  8700 ± 1700 200 ± 30 193 20HJ 1400± 400 2500 ± 600  48,000 ± 7000  >11,000 194 20HK 195 20HL 1400 ± 200390 ± 120 21,000 ± 6000  4300 ± 1200 196 20HM  840 ± 100 450 ± 12080,000 ± 5000  >9,200 197 20HN 950 ± 70 500 ± 100 9500 ± 400 11,300 ±100   198 20HO  700 ± 100 2800 ± 600  >8,200 >82,000 199 20HP 4700 ± 6002500 ± 1100 >93,000 >9,300 200 20HQ 201 20HR  360 ± 110 260 ± 70  13,000± 1700  26,000 ± 7000  202 20HS 1200 ± 100 650 ± 100 >62,000 >6200 20320HT  760 ± 170 940 ± 330 48,000 ± 6000  >5500 204 20HU 220 ± 40 1600 ±300  4100 ± 800 8600 ± 800 205 20HV  8400 ± 2200 1200 ±400  >185,000 >2,600 206 20HW 610 ± 40 230 ± 20  20,000 ± 1000  8200 ±200 207 20HX 570 ± 60 410 ± 60  208 20HY 1200 ± 100 930 ± 160 25,000 ±3000  209 20HZ  3900 ± 1100 610 ± 100 >90,000 210 20IA 40,000 ± 4000 5500 ± 600  211 20IB  470 ± 120 430 ± 100 59,000 ± 9000  212 20IC  80 ±10 55 ± 5  213 20ID 2300 ± 700 1700 ± 700  214 20IE 2900 ± 800 360 ± 30 215 20IF 26,000 ± 3000  490 ± 120 216 20IG 460 ± 60 150 ± 40  217 20IH2,200 ± 100  2,200 ± 100   43,000 ± 4,000 >7,000 218 20II 10,000 ± 3,000600 ± 200 15,000 ± 6,000  600 ± 100 21920IJ >52,000 >52,00 >52,000 >52,000 220 20IK  90 ± 10 130 ± 10  101,000± 18,000 40,000 ± 3,000 221 20IL >21,000 2,500 ± 200   >21,000 >21,000222 20IM 5,000 ± 100  1,100 ± 100   9,300 ± 200  330 ± 60 223 20IN20,000 ± 3,700 2,700 ± 300   >185,000 >55,000 22420IO >200,000 >130,000 >200,000 >130,000 225 20IP 47,000 ± 4,000 55,000± 11,000 >350,000  33,000 ± 13,000 22620IQ >59,000 >59,000 >59,000 >59,000 227 20IR >57,000 4,400 ±700   >57,000 16,000 ± 4,000 228 20IS >38,000 >38,000 24,000 ±3,000 >38,000 229 20IT >56,000 >2,000 >56,000 >2,000 230 20IU 620 ± 80100 ± 10  38,000 ± 5,000 1,000 ± 200  231 20IV 1,500 ± 100  280 ± 10 14,000 ± 4,000 232 20IW  700 ± 100 460 ± 60  42,000 ± 6,000 3,300 ± 600 233 20IX 3,200 ± 300  350 ± 80  >150,000 2,400 ± 700  234 20IY 3,000 ±300  1,100 ± 400   24,000 ± 6,000  9,000 ± 1,000 235 20IZ 3,500 ± 400 2,200 ± 400   49,000 ± 6,000  6,500 ± 1,600 236 20JA 49,000 ± 11,00029,000 ± 5,000   48,000 ± 10,000 237 20JB 1,200 ± 300  730 ± 140 22,000± 1,000 6,600 ± 900  238 20JC  780 ± 190 57 ± 8  23,000 ± 2,000  4,700 ±1,200 239 20JD 420 ± 60 70 ± 20 39,000 ± 3,000 28,000 ± 4,000 240 20JE2,900 ± 100  1,300 ± 200   >24,000 1,300 ± 100  241 20JF 560 ± 90 110 ±20  >28,000 18,000 ± 4,000 242 20JG 2,400 ± 400  580 ± 150 18,000 ±2,000 2,900 ± 600  243 20JH 2,200 ± 500  670 ± 240  64,000 ± 10,00026,000 ± 6,000 244 20JI 1,600 ± 400  150 ± 10   87,000 ± 11,000 35,000 ±7,000 245 20JJ  3,400 ± 1000  440 ± 90  79,000 ± 7,000 14,000 ± 1,700246 20JK 2,800 ± 260  1,900 ± 450   14,000 ± 2,000  6,200 ± 1,300 24720JL  6,100 ± 2,000 1,200 ± 250   10,000 ± 1,400  7,100 ± 1,700 248 20JM 830 ± 100 200 ± 25  23,000 ± 1,000  610 ± 120 249 20JN 4,100 ± 820  420± 100 18,000 ± 3,500 19,000 ± 3,800 250 20JO  99,000 ± 21,000 137,000 ±14,000  >275,000 137,000 ± 10,000 251 20JP 128,000 ± 4,000  51,000 ±1,000  >275,000 82,000 ± 8,000 252 20JQ >380,000 33,000 ±3,000  >380,000 >380,000 253 20JR >380,000 >38,000 >380,000 >380,000 25420JS 2,700 ± 800  1,100 ± 100   43,000 ± 6,000 >65,000 255 20JT 2,900 ±500  55 ± 2  119,000 ± 15,000 99,000 ± 4,000 256 20JU 1,500 ± 200  880 ±200 7,500 ± 800  7,100 ± 300  257 20JV 2,800 ± 600  320 ± 30  25,000 ±2,000 26,000 ± 3,000 258 20JW >45,000 >45,000 >45,000 >45,000 259 20JX16000 ± 3000 2400 ± 200  >85,000 4700 ± 400 260 20JY 1600 ± 500 150 ±20  >64,000 19000 ± 4500 261 20JZ  6300 ± 1100 1000 ± 150  64000 ± 200038000 ± 2100 262 20KA  8700 ± 1300 3900 ± 570  287000 ± 14000  73000 ±17000 263 20KB 2000 ± 300 1400 ± 200  124000 ± 18000 39000 ± 7000 26420KC 1400 ± 100 76 ± 17 >85,000  54000 ± 20000 265 20KD 810 ± 20 8 ± 11100 ± 200 250 ± 80 266 20KE 140 ± 20 70 ± 18  56000 ± 15000 32000 ±7000 267 20KF  900 ± 160 160 ± 20  >90,000 28000 ± 8000 268 20KG 2100 ±200 330 ± 90  54,000 ± 16000  >8,000 269 20KH 11000 ± 3000 850 ± 32052000 ± 4000 >7,000 270 20KI  8000 ± 1500 1300 ± 100  >84,000 7100 ± 700271 20KJ 1700 ± 200 200 ± 90  >93,000 >9,300 27220KK >46,000 >4,700 >47,000 >4,700 273 20KL 30000 ±5000 >1,500 >45,000 >4,500 274 20KM 39000 ± 3000 1200 ±300  >46,000 >4,500 275 20KN 1500 ± 300 370 ± 40  >62,000 >6,200 27620KO 1500 ± 200 760 ± 100 >61,000 >6,100 277 20KP 760 ± 70 190 ± 20 31,000 ± 6000  9,800 ± 1000  278 20KQ 1000 ± 100 270 ± 10  >94000 >9,400279 20KR 1700 ± 400 190 ± 20  >90000 >9,000 280 20KS 2400 ± 800<80 >83000 >2,800 281 20KT 1800 ± 700 170 ± 10  27000 ± 2000 5000 ± 700282 20KU 680 ± 60 110 ± 10  >85000 >8,500 283 20KV  2900 ± 1200 300 ±20  40000 ± 4000 >9,300 284 20KW 13,600(N = 2) 340 ± 20  >8,800 285 20KX 3800 ± 1100 310 ± 20  84000 ± 9000 2000 ± 100 286 20KY  48000 ± 100006300 ± 200  51000 ± 1700 >8,800 287 20KZ 455000 ± 22000 1100 ± 100 567000 ± 17000 4700 ± 400 288 20LA 1800 ± 600 150 ± 20  11000 ± 3200~9,000 289 20LB 51 ± 4 530 ± 150 >290000 >8,800 290 20LC 291 20LD 29220LE 293 20LF 294 20LG  960 ± 170 295 20LH 200 ± 44 296 20LI  250 (N =2) 297 20LJ 2200 (N = 1) 298 20LK >7000 299 20LL 300 20LM 301 20LN 30220LO 303 20LP 304 20LQ 305 20LR 306 20LS 307 20LT 308 20LU 309 20LV 31020LW 311 20LX 312 20LY 313 20LZ 314 20MA 315 20MB 316 20MC

What is claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein X and R₁ are connected through a Linker Unit and X is selected from the group consisting of —NH—, —NR₈—, —S—, —O—, —CH₂—, —CHR₈—, —CHR₈R₉—, —OC(═O)—, —N(R₃)S(═O)—, and —N(═N)—; R₁ represents an Affinity Moiety selected from the group consisting of an antibody, an antibody fragment, a receptor protein, a peptidic growth factor, an anti-angiogenic protein, a specific binding peptide, a protease cleavable peptide, a glycopeptide, a peptide, a natural peptidic toxin, a protein toxin and an oligonucleotide; R₃, R₄, R₅, R₆, and R₇ each independently represent —H, -, —OH, —OCH₃, —OC(═O)CH₃ —CH₃, —CH₂—CH₃, —CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃,—CH₂OH, and —(C₁-C₄)alkyl; R₈, and R₉ each independently represent —CH₂—, -, —CH₂—CH₂—, —CH(CH₃)—CH₂—, —C(CH₃)₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH(CH₃)—CH₂—, —CH—C(CH₃)₂—CH₂—, —C(═O)CH₂—, —CH₂O—; where the Linker Unit is selected from the group consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl.
 2. The compound of claim 1, where X represents O; R₃, R₅, R₆, R₇ ═CH₃; and R₄ ═H.
 3. The compound of claim 1, where X represents O; R₃, R₅, R₆ ═CH₃; R₄ ═H; and R₇ =CH₂OH.
 4. The compound of claim 1, where the Affinity Moiety comprises a molecule with chemical groups selected from the group consisting of primary amino groups and hydroxyl groups or combinations thereof.
 5. A composition comprising racemic mixtures of the compound of claim
 1. 6. A composition comprising enantiomers of the compound of claim
 1. 7. A composition comprising enantiomers and racemic mixtures of the compound of claim
 1. 8. A composition comprising the compound of claim 1 and a physiologically compatible excipient.
 9. The composition of claim 8 forming a medicant.
 10. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein X and R₁ are connected through a Linker Unit; where X is selected from the group consisting of a secondary amino group, a carbonyl group, an ester, and an alcohol; R₁ represents an Affinity Moiety selected from the group consisting of an antibody, an antibody fragment, a receptor protein, a peptidic growth factor, an anti-angiogenic protein, a specific binding peptide, protease cleavable peptide, a glycopeptide, a peptide, a natural peptidic toxin, a protein toxin and an oligonucleotide; R₃, R₄, R₅, R₆, R₇ each independently represent —H, -, —CH₃, —CH₂—CH₃, —CH—(CH₃)₂, —C(CH₃)₃, —CH₂—CH₂—CH₃, —CH₂—CH—(CH₃)₂, —CH—C—(CH₃)₃, —C(═O)CH₃, —CH₂OH, and —(C₁-C₄)alkyl; where the Linker Unit is selected from the group consisting of 4-fluorosulfonyl benzoyl, 3-fluorosulfonyl benzoyl and 2-fluorosulfonyl benzoyl.
 11. A composition comprising racemic mixtures of the compound of claim
 10. 12. A composition comprising enantiomers of the compound of claim
 10. 13. A composition comprising enantiomers and racemic mixtures of the compound of claim
 10. 14. A composition comprising the compound of claim 10 and a physiologically compatible excipient.
 15. The composition of claim 14 forming a medicant. 